Wellesley CollegeWellesley College Digital Scholarship and Archive

Honors *esis Collection

2016

Optimizing the apFRET technique to investigatethe interaction of the cardiac potassium channelshERG and KvLQT1Heidi Wade

Follow this and additional works at: h+p://repository.wellesley.edu/thesiscollection

*is *esis is brought to you for free and open access by Wellesley College Digital Scholarship and Archive. It has been accepted for inclusion inHonors *esis Collection by an authorized administrator of Wellesley College Digital Scholarship and Archive. For more information, please contactir@wellesley.edu.

! 1!

Optimizing)the)apFRET)technique)to)investigate)the)interaction)of)the)cardiac)potassium)channels)hERG)and)KvLQT1))!!

Heidi!M.!Wade,!Louise!E.O.!Darling!

Biochemistry!Department!Wellesley!College!

!

Honor!Thesis!2016!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

! 2!

!

Table&of&Contents&!!

Abbreviations………………………………………………………………………………….4)Abstract………………………………………………………………………………………….5)Introduction……………………………………………………………………………………6)

hERG)(KCNH2))KvLQT1)(KCNQ1))Motivation))Scientific)Approach))

Methods)and)Materials….……………………………………………………………19)HEK)293)Cell)Culture,)Transfection,)and)Fixation)apFRET)Experimentation)

Results…………………………………………………………………………………………23)Optimization)of)apFRET)experimental)parameters)Acceptor)(YFP))detection)range)Donor)(CFP))detection)range)Control)apFRET)experiments))Experimental)apFRET)experiments))

Discussion)and)Future)Directions…………………………………………………51)References……………………………………………………………………………..……59)

))))!!!!!!!!

!

! 3!

!Acknowledgments:///I!would!like!to!thank!the!following!people!who!helped!me!in!the!completion!of!this!thesis.!Firstly,!my!advisor!Louise!Darling!for!all!of!her!assistance!and!mentorship!over!the!past!two!years.!Secondly,!the!currents!members!of!the!Darling!lab!including!Laurel!Kinman,!Medeea!Popescu,!Myfanwy!Adams,!and!Stephanie!Kim!and!former!members!Estelle!Kim!(’15)!and!Yeon!Joo!Lee!(’15).!Thirdly,!my!thesis!advisors!Alex!Diesl,!Don!Elmore,!and!Vanja!KlepacOCeraj!for!their!mentorship!and!support.!Finally,!all!of!the!Wellesley!College!Biochemistry!faculty!for!their!teaching!and!mentorship!during!my!time!at!Wellesley.!!!!!!!///////////////////////////////

! 4!

Abbreviations://!apFRET:!acceptor!photobleach!Förster!Resonance!Energy!Transfer//cAMP:!cyclic!adenosine!monophosphate!!CHO:!Chinese!hamster!ovary!!!CFP:!cyan!fluorescent!protein!!FRET:!Förster!Resonance!Energy!Transfer!IBMX:!3OisobutylO1Omethylxanthine,!a!phoshodiesterase!inhibitor!that!prevents!breakdown!of!cAMP!IKr:!rapidly!activating!component!of!the!delayed!rectifier!current!!IKs:!slowly!activating!component!of!the!delayed!rectifier!current!!INA:!sodium!current!!HEK:!human!embryonic!kidney!!hERG!and!hERG:!human!etherOaOgoOgo!related!gene!and!its!protein!product,!respectively;!pore!forming!αOsubunit!of!a!voltage!gated!potassium!channel!hERG!!KCNH2:!gene!encoding!for!hERG,!alternative!name!to!hERG!KCNE1:!gene!encoding!for!minK!KCNQ1:!gene!encoding!for!KvLQT1!KvLQT1:!!αOsubunit!of!a!voltage!gated!potassium!channel!KvLQT1!LQTS:!Long!QT!syndrome!minK:!minimal!potassium!channel!subunit;!βOsubunit!known!to!associate!with!hERG!and!KvLQT1!channels!MiRP1:!minKOrelated!peptide!1;!βOsubunit!proposed!to!associate!with!hERG!ROI:!region!of!interest!YFP:!yellow!fluorescent!protein!/////////////////////

! 5!

/Abstract://hERG!and!KvLQT1!are!voltageOgated!potassium!channel!protein!αOsubunits!encoded!for!by!KCNH2!and!KCNQ1,!respectively,!that!play!a!critical!role!in!regulating!the!human!heartbeat.!hERG!and!KvLQT1!respectively!produce!the!rapidly!activating!(IKr)!and!slowly!activating!(IKs)!components!of!the!cardiac!repolarization!current,!which!plays!a!critical!role!in!regulating!the!action!potential!duration! in!cardiomyocytes.!Both!hERG!and!KvLQT1!channels!have!been!shown!to!associate!with!the!βOsubunit,!minK.!While!the!expression!of!minK!is!not!necessary!for! the! production! of! IKr,! the! association! of! minK! with! KvLQT1! is! essential! for! the! full!production!of!IKs.!Previously!performed!biochemical!assays!and!acceptor!photobleach!FRET!(apFRET)! experiments! indicate! that! hERG! and! KvLQT1! physically! interact! via! their! –C!termini.!The!Darling!lab!has!characterized!the!interaction!of!hERG!and!KvLQT1!via!apFRET!experiments!and!found!there!to!be!a!positive!FRET!efficiency!for!the!hERGOmCFP!+!KvLQT1OmYFP! pair.! This! thesis! aimed! to! investigate! whether! interchanging! the! fluorophores!attached!to!hERG!and!KvLQT1!affects!the!FRET!efficiency.!In!addition,!this!work!addresses!whether!the!concatenation!of!minK!to!KvLQT1,!so!the!construct!can!produce!full!IKs!current,!alters! the! interaction! between! KvLQT1! and! hERG,! as!measured! via! apFRET.! In! order! to!address!these!questions,!apFRET!experiments!were!performed!on!KvLQT1OmCFP!+!hERGOmYFP!and!hERGOmCFP!+!KvLQT1OminKOmYFP!FRET!pairs.!It!was!found!that!the!mean!FRET!efficiencies!of!KvLQT1OCFPOYFP!(positive!control,!n=24),!hERGOCFP!(negative!control,!n=30),!hERGOCFP!+!KvLQT1OYFP!(experimental!control,!n=24)!were!27.6%!±!7.2%,!O0.7%!±!6.6%,!0.3%! ±5.6%! respectively.! The! mean! FRET! efficiencies! for! the! KvLQT1OCFP! +! hERGOYFP!(n=15)! and! hERGOCFP! +! KvLQT1OminKOYFP! pairs!were! O0.1%! ±! 4.9%! and! O2.1%! ±! 6.1%!(n=20).!The!observed!FRET!efficiency!close!to!0%!for!the!experimental!control,!hERGOCFP!+!KvLQT1OmYFP,!is!an!unexpected!result!based!on!previously!published!and!ongoing!work!in!the! Darling! lab.! Thus,! these! data! suggest! that! the! apFRET! technique! needs! further!optimization! before! evaluating! the! FRET! efficiencies! of! the! KvLQT1OCFP! +! hERGOYFP!and!hERGOCFP!+!KvLQT1OminKOYFP!FRET!pairs.!!

!

!

!

!

/

/

/

/

! 6!

Introduction:/

The!mechanical!function!of!the!heart!is!triggered!by!the!electrical!activity!of!

individual!heart!cells,!called!cardiomyocytes.!The!electrical!activity!within!cardiomyocytes!

is!generated!by!the!influx!of!sodium!ions!(Na+)!into!the!cell,!referred!to!as!the!sodium!

current!(INA),!and!efflux!of!potassium!ions!(K+)!from!the!cell,!referred!to!as!the!potassium!

current!(IK+).!However,!the!movement!of!other!ions!such!as!calcium!(Ca2+)!and!chloride!(ClO!

)!also!play!a!role!generating!the!electrical!activity!within!cardiomyocytes.!These!ions!

predominantly!flow!through!voltageOgated!channels!on!the!surface!of!the!cell!membrane,!

and!the!consequent!change!in!membrane!potential!that!results!from!the!movement!of!these!

ions!across!the!membrane!is!recognized!as!the!cardiac!action!potential!at!the!cellular!level,!

and!the!electrocardiogram!(EKG/ECG)!at!the!organismal!level.!!

The!standard!model!of!the!cardiac!action!potential!has!five!distinct!phases!(Figure!

1).!Phase!four!is!the!resting!membrane!potential!and!is!maintained!by!the!selective!

membrane!permeability!of!predominantly!K+!ions.!Phase!0!is!the!rapid!depolarization!

phase,!in!which!Na+!channels!rapidly!open!causing!a!rapid!influx!of!Na+!ions!into!the!cell!

(INA!rapid).!Phase!1!involves!a!partial!inactivation!of!the!Na+!channels,!and!the!flow!of!K+!

and!ClO!ions!across!the!membrane,!which!causes!a!small!downward!deflection!in!the!

membrane!potential.!Phase!2!is!the!plateau!phase!of!the!cardiac!action!potential!which!is!

predominantly!caused!by!a!balance!in!the!influx!of!Ca2+!!ions!(ICaOL)!and!the!efflux!of!K+!ions!

through!the!slow!component!of!the!delayed!rectifier!current!(IKS).!Phase!3!is!the!rapid!

repolarization!phase,!in!which!the!Ca2+!channels!close!and!the!slow!delayed!K+!rectifier!

channels!remain!open,!causing!a!net!positive!outward!current!which!causes!the!membrane!

potential!to!become!more!negative.!As!the!membrane!potential!becomes!more!negative,!

! 7!

additional!K+!channels!open,!such!as!the!rapid!K+!delayed!rectifier!channels!(IKr),!through!

which!additional!K+!ions!flow.!!The!efflux!of!K+!ions!during!this!phase!results!in!

repolarization!of!the!cell!to!resting!membrane!potential,!which!marks!the!end!of!the!

heartbeat.!

!

Figure/1./Standard/model/of/the/five/phases/of/the/cardiac/action/potential.!During!each!of!the!five!phases!various!ions!(blue)!flow!through!membrane!channels!(green)!causing!a!net!change!in!the!membrane!potential.!The!most!prominent!currents!associated!with!the!phases!are!indicated!in!black!text.!Image!adapted!from!Sherwood!2008.!!!

While!only!a!single!sodium!current,!that!is!conducted!by!a!single!sodium!channel!

protein,!is!responsible!for!depolarization,!there!are!at!least!ten!different!potassium!

currents!regulated!by!at!least!twelve!potassium!channel!proteins!that!are!responsible!for!

repolarization!(Grant,!2009).!The!delayed!rectifier!current!is!one!of!the!multiple!potassium!

currents!responsible!for!the!repolarization!that!occurs!during!phases!two!and!three!of!the!

action!potential.!The!delayed!rectifier!current!is!made!up!of!the!rapidly!activating!(IKr)!and!

slowly!activating!(IKs)!currents!which!are!respectively!produced!by!the!voltageOgated!

potassium!channels!hERG!and!KvLQT1.!Proper!regulation!of!the!delayed!rectifier!current!is!

essential!for!maintaining!a!normal!heartbeat.!Consequently,!loss!of!function!mutations!or!

! 8!

blockage!of!either!hERG!or!KvLQT1!can!result!in!a!variety!of!cardiac!diseases!and!

arrhythmias!such!as!the!heritable,!genetic!condition!Long!QT!syndrome!(LQTS).!

Specifically,!loss!of!function!mutations!of!KCNQ1,!the!gene!encoding!KvLQT1,!and!KCNH2,!

the!gene!encoding!HERG,!account!for!more!than!80!percent!of!autosomal!dominant!LQTS!

(Grant,!2009). Brief!descriptions!of!hERG!and!KvLQT1!are!outlined!in!the!following!two!

sections.

hERG!(KCNH2):!

The!poreOforming!αOsubunit!of!the!hERG!channel!that!produces!the!IKr!component!of!

the!delayed!rectifier!current!is!encoded!for!by!KCNH2!located!on!chromosome!seven.,In,

vivo,!hERG!functions!as!a!homotetramer!with!the!pore!domain!from!each!of!the!four!αO

subunits!forming!a!central!cavity!that!can!conduct!ions!(Curren!et!al.,!1995).!!As!a!

hemotetramer,!hERG!is!a!1,159!amino!acid!residue!protein!that!contains!six!

transmembrane!domains!(S1OS6)!(Warmke!et!al.,!1994).!hERG!contains!an!intracellular!ON!

terminus!with!a!PerOArntOSim!(PAS)!domain!that!is!characteristic!of!!the!etherOaOgoOgo!

subfamily!of!!voltageOgated!K+!channels!and!an!intracellular!OC!terminus!that!contains!a!

cyclic!nucleotide!binding!homology!domain!(Cabral!et!al.,!1998)(Warmke!et!al.,!1994).!In,

vivo,!hERG!is!capable!of!assembling!with!the!βOsubunits!minimal!potassium!channel!

subunit!(minK),!encoded!for!by,KCNE1,!and!minKOrelated!peptide!1!(MiRP1),!which!is!

encoded!KCNE2!(Anantharam,!2005).!However,!despite!the!reported!association!of!these!βO

subunits,!their!function!remains!unknown.!It!appears!MiRP1!alters!hERG!current!density!

and!gating,!while!the!association!of!minK!has!no!such!effect!on!hERG’s!functionality!

(Anantharam,!2005).!!

! 9!

Figure/2./Structure/of/hERG./(A)!depicts!a!linear!sequence!of!the!hERG!channel!αOsubunit,!with!crucial!domains!colorOcoded.!(B)!depicts!an!extracellular!(right)!and!intraceullular!top!view!(left)!of!hERG!tetramers,!and!(C)!depicts!a!membrane!imbedded!view!of!hERG!with!two!αOsubunits!shown./Image!taken!from!Vandenberg!2012.!!!KvLQT1!(KCNQ1):!

The!αOsubunit!of!the!KvLQT1!channel,!which!contributes!to!the!IKs!component!of!the!

delayed!rectifier!current!and!is!associated!with!phases!two!and!three!of!the!cardiac!action!

potential,!is!encoded!for!by,KCNQ1,!located!on!chromosome!11.!KvLQT1!is!a!676!amino!

acid!residue!protein!that!contains!six!transmembrane!domains!and!intracellular!ON!and!OC!

termini!(Yang,!1997).!In,vivo,!KvLQT1!functions!as!a!homotetramer!and!assembles!with!the!

βOsubunit!minK!encoded!for!by!KCNE1,!located!on!chromosome!21.!Unlike!hERG!which!

does!not!need!expression!of!the!βOsubunit!minK!in!order!to!produce!IKr!,!expression!of!

minK!is!necessary!for!production!of!proper!IKS!current.!!Splawski!et!al.!showed!that!

coexpression!of!minK!with!KvLQT1!resulted!in!an!increase!in!the!current,!a!positive!shift!in!

the!voltage!activation!threshold,!slowing!of!activation!and!deactivation,!and!almost!

! 10!

complete!absence!of!inactivation!(Splawski!et!al.,1997).!Specifically,!a!fourO!to!sevenOfold!

increase!in!the!channel!conductance!of!KvLQT1!has!been!shown!upon!association!with!

minK!(Pusch!1998,!Sesti!1998,!Yang!1998).!The!ratio!of!minK:!hERG!subunits!has!not!been!

confirmed,!but!it!is!generally!believed!that!proteins!assemble!in!a!1:2!ratio!(Sesti!1998).!!

!

Figure/3./Structure/of/KvLQT1.!(A)!depicts!a!membrane!topology!diagram!of!a!KvLQT1!αOsubunit!along!with!minK!and!(B)!depicts!KvLQT1!as!a!transmembrane!homotetramer!with!minK!associated.!Image!taken!from!Jespersen,!2005.!!!!Motivation:!!!

Why!there!are!multiple!potassium!currents,!regulated!by!multiple!potassium!

channels,!yet!only!a!single!sodium!current,!regulated!by!a!single!potassium!channel,!within!

cardiomyocytes!has!been!a!question!within!the!scientific!community.!Historically,!it!was!

assumed!that!the!excess!of!potassium!currents!were!acting!as!a!“repolarization!reserve”,!

meaning!if!there!was!a!block!or!a!mutation!in!one!potassium!channel!that!rendered!it!to!

! 11!

have!reduced!or!nonOfunction,!then!the!other!potassium!channels!could!compensate!for!

this!through!increased!activity!(Roden!et!al.,!2008).!However,!the!idea!that!excess!

potassium!channels!are!acting!as!a!“repolarization!reserve”!has!been!challenged!by!

research!showing!that!loss!of!either!IKs!or!IKr!in!a!transgenic!rabbit!model!resulted!in!the!

mutual,!functional!downregulation!of!the!respective!complementary!potassium!current,!IKr!

or!IKs!(Brunner!et!al.,!2008).!The!observation!of!a!mutual,!functional!downregulation!of!IKr!

and!IKs!was!further!confirmed!in,vitro.!Ren!et!al.!(2010)!showed!that!Chinese!Hamster!

Ovary!(CHO)!cell!lines!stably!expressing!KvLQT1OminK!and!then!transiently!transfected!

with!expression!plasmids!encoding!for!mutant!or!wildOtype!HERG!downregulated!IKs!

without!affecting!the!steadyOstate!expression!level!of!KvLQT1.!Similarly,!they!observed!that!

CHO!cells!stably!expressing!hERG!and!transiently!transfected!with!expression!plasmids!

encoding!for!mutant!or!wildOtype!KvLQT1OminK!downregulated!IKr!without!affecting!the!

steady!state!expression!level!of!hERG!(Ren!et!al.,!2010).!!

The!observed!mutual,!functional!downregulation!of!IKr!and!IKs!suggested!that!

interactions!between!hERG!and!KvLQT1!are!important!for!proper!IKr!and!IKs!(Brunner!et!al.,!

2008;!Ren!et.!al.,!2010).!Ren!et!al.!(2010)!showed!that!hERG!and!KvLQT1!physically!

interact!via!coOimmunoprecipitation!assays!and!showed!that!there!is!a!direct!association!

between!the!–C!termini!of!KvLQT1!and!hERG!using!surface!plasmon!resonance!imaging!

(Ren!et!al.,!2010).!This!finding!supported!the!work!of!Ehrlich!et!al.!(2004)!which!showed!

that!hERG!and!KvLQT1!coOimmunioprecipitated!in!cultured!CHO!cells!as!well!as!in!native!

cardiac!tissue,!and!that!KvLQT1!bound!to!a!–C!terminus!hERG!glutathione!SOtransferase!

fusion!protein!in!a!protein!interaction!assay!(Ehrlich!et!al.,!2004).!!

! 12!

Additionally,!florescence!microscopy!assays!have!provided!evidence!of!the!

interaction!between!hERG!and!KvLQT1!in!an!intact!cellular!environment.!OrganODarling!et!

al.!(2013)!used!acceptor!photobleach!Förster!Resonance!Energy!Transfer!(apFRET)!to!

show!that!hERG!and!KvLQT1!interact!within!the!heterologous!human!embryonic!kidney!

293!(HEK!293)!cell!line!and!primary!neonatal!rabbit!cardiomyocytes.!apFRET!experiments!

showed!the!largest!FRET!efficiencies!between!KvLQT1!and!hERG!subunits!that!were!–C!

terminally!labeled!with!the!fluorophores,!as!opposed!to!ON!terminally!labeled,!supporting!

the!observation!that!the!two!proteins!interact!via!their!–C!termini.!!Furthermore,!OrganO

Darling!et!al.!(2013)!demonstrated!that!acute!treatment!with!a!membraneOpermeable!

cAMP!analog!+!IBMX!reduced!the!extent!of!interactions!between!hERG!and!KvLQT1.!IBMX!

is!a!phosphodiesterase!inhibitor!that!prevents!the!breakdown!of!cAMP,!and!together!with!

the!cAMP!analog!elevates!intracellular!cAMP!levels.!The!fact!that!interactions!between!

hERG!and!KvLQT1!were!reduced!in!the!presence!of!the!membraneOpermeable!cAMP!analog!

+!IBMX!suggests!that!the!interaction!of!hERG!and!KvLQT1!is!regulated!by!intracellular!

cAMP!levels.!!

Despite!the!confirmation!of!hERG!and!KvLQT1’s!interaction!via!classical!

biochemical,!biophysical,!and!florescence!microscopy!methods,!the!specific!molecular!

mechanisms!underlying!their!interaction!remain!poorly!understood.!For!example,!while!it!

is!generally!assumed!that!the!proteins!interact!via!their!–C!termini!the!specific!amino!acid!

residues!necessary!for!their!interaction!have!yet!to!have!been!identified.!Furthermore,!it!is!

not!known!whether!hERG!and!KvLQT1!interact!on!the!plasma!membrane!or!intracellularly!

during!biogenesis,!such!as!in!the!endoplasmic!reticulum.!Additionally,!the!mechanism!of!

how!cAMP!modulates!the!interaction!between!KvLQT1!and!hERG!has!not!been!established.!

! 13!

Within!the!Darling!lab,!we!are!focused!on!elucidating!some!of!these!fundamental!questions!

about!the!mechanism!of!interaction!between!hERG!and!KvLQT1.!Currently,!we!are!most!

interested!in!determining!the!role!of!cAMP!in!modulating!the!interaction,!and!where!in!the!

cell!hERG!and!KvLQT1!interact.!One!way!we!investigate!the!interaction!between!hERG!and!

KvLQT1!in!order!to!answer!these!questions!is!through!the!use!of!the!quantitative!

florescence!microscopy!technique!apFRET.!!

The!apFRET!method!takes!advantage!of!the!naturally!occurring!FRET!phenomenon,!

which!involves!a!nonOradiative!transfer!of!energy!between!an!“acceptor”!and!“donor”!

fluorophore!(Figure!4A).!!In!order!for!FRET!to!occur!the!donor!fluorophore!must!have!an!

emission!spectrum!that!overlaps!with!the!excitation!spectrum!of!the!acceptor!fluorophore.!

For!this!reason,!Cyan!Florescent!Protein!(CFP)!(emission!max=485nm)!is!commonly!used!

as!the!donor!with!Yellow!Florescent!Protein!(YFP)!(excitation!max=527)!as!the!acceptor!

(Figure!8D).!In!addition,!the!two!fluorophores!must!be!less!than!10!nm!apart!for!FRET!to!

occur!(Piston,!2007).!Consequently,!FRET!can!be!used!to!indirectly!detect!if!two!proteins!

are!likely!interacting!by!conjugating!each!of!the!two!proteins!of!interest!with!either!the!

donor!(CFP)!or!acceptor!(YFP)!fluorophore!and!measuring!the!FRET!efficiency!that!occurs!

(Figure!4B).!

! 14!

Figure/4./FRET/involves/a/nonIradiative/transfer/of/energy/from/a/donor/fluorophore/to/an/acceptor/fluorophore.!(A)!depicts!a!Jablonski!diagram!representing!the!energy!exchange!characteristic!of!FRET.!In!FRET,!the!donor!molecule!absorbs!light!that!excites!its!electrons!to!a!higher!energy!state.!Instead!of!falling!back!down!to!the!lower!energy!state!and!emitting!fluorescence,!the!energy!is!transferred!to!excite!the!electrons!in!the!acceptor!molecule!to!a!higher!energy!state.!The!electrons!then!fall!back!down!to!their!lower!energy!state!emitting!fluorescence.!(B)!depicts!a!scheme!for!how!FRET!can!be!used!to!detect!the!physical!interaction!of!two!proteins.!If!a!donor!and!an!acceptor!molecule!are!conjugated!to!the!two!proteins!of!interest,!and!if!those!proteins!are!in!close!physical!proximity,!FRET!will!occur!and!fluorescence!emission!from!the!acceptor!molecule!will!be!detected!as!illustrated!in!the!rightmost!image.!If!the!proteins!are!not!in!close!physical!proximity,!FRET!will!not!occur!and!fluorescence!emission!from!the!donor!molecule!will!be!detected.!!!

The!apFRET!technique!has!the!distinct!advantage!of!allowing!one!to!quantitatively!

study!the!interaction!of!two!proteins!in!an!intact!cellular!environment.!In!this!way!in!it!

superior!to!classical!biochemistry!approaches!used!to!study!proteinOprotein!interactions,!

such!as!coOimmunoprecipation!“pull!down”!assays,!which!require!lysing!of!the!cell.!Despite!

this!advantage,!one!of!the!limitations!of!this!method!(and!all!light!microscopy!techniques)!

is!that!there!is!uncertainty!regarding!whether!the!population!of!hERG!and!KvLQT1!proteins!

being!measured!are!located!at!the!plasma!membrane!and!thus!fully!capable!of!conducting!

the!proper!potassium!currents.!For!instance,!a!subOpopulation!of!proteins!being!measured!

with!apFRET!may!reside!below!the!surface!of!the!cell!membrane!where!it!is!unable!to!

facilitate!the!movement!of!potassium!ions!across!the!cell!membrane.!!

! 15!

This!constraint!is!limiting!because!in!order!to!address!the!question!of!how!hERG!and!

KvLQT1!physically!interact!to!regulate!the!potassium!current,!it!is!essential!that!the!

interaction!of!hERG!and!KvLQT1!be!measured!in!fully!functional!proteins.!One!approach!to!

ensure!that!the!physical!interaction!of!hERG!and!KvLQT1!is!being!measured!primarily!from!

functional!forms!of!these!proteins!is!to!perform!electrophysiology!experiments!in!parallel!

with!apFRET!experiments.!Electrophysiology!experiments!measure!the!current!conducted!

by!channel!proteins!and!thus!can!be!used!to!verify!that!there!is!current!flowing!through!the!

populations!of!hERG!and!KvLQT1!channels!selected!for!apFRET!experimentation.!However,!

performing!such!electrophysiology!experiments!(in!collaboration!with!John!Cameron’s!lab)!

requires!coOexpression!of!the!βOsubunit!minK,!whose!association!with!KvLQT1!is!necessary!

for!proper!IKs!formation.!We!have!taken!the!approach!of!creating!plasmids!that!express!

minK!and!KvLQT1!directly!concatenated!with!KvLQT1!coming!after!the!COterminus!of!

minK.!

The!Darling!lab!has!previously!shown!that!hERG!and!KvLQT1!interact!via!apFRET!

experiments!but!has!yet!to!determine!whether!the!same!interaction!is!observed!for!hERG!

and!KvLQT1!concatenated!with!minK.!Consequently,!the!primary!aim!of!my!thesis!is!to!

address!this!question!by!comparing!the!degree!of!physical!interaction!between!hERG!and!

KvLQT1!and!hERG!and!KvLQT1OminK!using!apFRET.!It!is!possible!that!linking!minK!to!

KvLQT1!affects!the!three!dimensional!conformation!of!KvLQT1!in!such!a!way!that!disrupts!

its!physical!interaction!with!hERG.!Thus,!before!utilizing!KvLQT1OminK!linked!constructs!in!

other!experiments,!it!is!necessary!to!verify!that!the!linkage!of!minK!to!KvLQT1!does!not!

disrupt!the!interaction!of!KvLQT1!and!hERG.!!

This!is!crucial!to!move!research!forward!within!the!Darling!lab!because!it!will!

! 16!

provide!verification!of!whether!insights!into!the!molecular!mechanisms!of!the!hERG!and!

KvLQT1!interaction!are!physiologically!relevant.!Concatenation!of!minK!to!KvLQT1!is!

necessary!for!proper!IKs!!formation;!consequently,!in!order!to!perform!patch!clamping!

experiments,!which!offer!insights!into!the!function!of!hERG!and!KvLQT1,!it!is!essential!that!

minK!is!concatenated!to!KvLQT1.!Addressing!this!question!is!important!because!

performing!apFRET!experiments!within!the!same!experimental!situation!as!patch!clamping!

experiments!will!allow!for!functional!and!interaction!data,!collected!via!apFRET,!to!be!

compared.!This!is!of!interest!to!the!Darling!lab!because!complementation!of!functional!

data,!collected!via!patch!clamping,!and!interaction!data,!collected!via!apFRET!experiments,!

will!provide!a!clearer!picture!of!the!mechanism!of!interaction!between!hERG!and!KvLQT1,!

such!as!how!the!two!proteins!are!dynamically!regulated!over!time!or!regulated!by!

intracellular!changes!in!cAMP!levels.!

A!secondary!aim!of!my!thesis!is!to!address!whether!interchanging!the!fluorophores!

that!hERG!and!KvLQT1!are!tagged!with!affects!the!FRET!efficiency!between!the!two!

proteins.!The!Darling!lab!has!previously!measured!a!positive!FRET!efficiency!for!hERG!and!

KvLQT1!–C!terminally!labeled!with!CFP!and!YFP!respectively,!but!to!date!no!one!has!

investigated!whether!labeling!hERG!with!YFP!and!KvLQT1!with!CFP!yields!a!different!FRET!

efficiency.!It!is!not!expected!that!switching!the!fluorophores!would!affect!the!FRET!

efficiency!as!it!is!assumed!that!the!addition!of!a!fluorescent!label!does!not!change!the!three!

dimensional!conformation!of!the!proteins!or!prohibit!their!interaction!.!Additionally,!if!the!

addition!of!the!CFP!or!YFP!label!did!in!fact!interfere!with!the!interaction!of!hERG!and!

KvLQT1!it!is!unlikely!that!switching!the!fluorophores!that!hERG!and!kVLQT1!are!tagged!

with!would!affect!the!FRET!efficiencies!because!CFP!and!YFP!are!structurally!very!similar,!

! 17!

and!are!thus!likely!to!affect!the!proteins!in!a!similar!manner.!Despite!the!fact!that!it!is!

unlikely!that!such!an!interchange!affects!the!interaction!of!hERG!and!KvLQT1,!the!Darling!

lab!has!been!consistently!asked!to!address!this!question!by!grant!and!peer!reviewers!and!

thus!is!interested!in!obtaining!these!data!for!the!sake!of!being!scientifically!rigorous.!!

!

Scientific!Approach:!

To!address!whether!interchanging!the!fluorophores!that!hERG!and!KvLQT1!are!

tagged!with!affects!the!interaction!of!KvLQT1!and!hERG,!HEK!293!cells!were!transfected!

with!hERGOYFP!+!KvLQT1OCFP!and!hERGOCFP!+!KvLQT1OYFP!(experimental!control).!To!

address!the!question!of!whether!the!concatenation!of!minK!to!KvLQT1!affects!the!

interaction!of!KvLQT1!and!hERG,!HEK!293!cells!were!transfected!with!hERGOCFP+KvLQT1O

minK!OYFP!and!hERGOCFP+KvLQT1OYFP!(experimental!control).!!HEK!293!cells!were!used!

because!of!their!fast!and!easy!reproducibility!and!maintenance,!as!well!as!their!high!

transfection!and!protein!production!efficiency!(Thomas,!2005).!!

Post!transfection,!apFRET!experiments!were!performed!in!fixed!cells!using!confocal!

microscopy!and!the!FRET!efficiency!obtained!for!the!experimental!and!control!groups!was!

compared.!As!mentioned!previously,!apFRET!allows!for!!quantification!of!the!extent!to!

which!FRET!is!occurring!between!two!molecules!and!can!therefore!provide!information!

about!whether!two!proteins!interact.!apFRET!involves!the!comparison!of!the!amount!of!

donor!fluorescence!before!and!after!destroying!the!acceptor!fluorophore!via!

photobleaching.!If!FRET!is!occurring!between!the!two!proteins!of!interest!then!there!

should!be!an!increase!in!donor!fluorescence!upon!destruction!of!the!acceptor!fluorophore.!

The!extent!to!which!FRET!occurs!between!the!two!molecules!can!be!determined!by!

! 18!

calculating!the!FRET!efficiency!using!the!following!equation:!FRETeff=!(DpostODpre)/Dpost,!

where!Dpost!is!the!fluorescence!intensity!of!the!donor!after!acceptor!photobleaching,!and!

Dpre!is!the!fluorescence!intensity!of!the!donor!before!acceptor!photobleaching.!!A!positive!

FRET!efficiency!indicates!proteinOprotein!interaction,!and!FRET!efficiencies!using!

fluorescent!proteins!as!fluorophores!usually!do!not!exceed!50!percent!due!to!the!physical!

constraint!that!the!fluorophore!portion!of!the!proteins!can!only!get!so!close!to!one!another!

(Piston,!2007).!A!general!scheme!describing!the!apFRET!method!is!outlined!in!figure!5.!!

!

Figure/5./The/apFRET/method/can/be/used/to/quantitatively/access/the/interaction/of/two/proteins.!apFRET!involves!labeling!two!proteins!of!interest!with!a!“donor”!fluorophore!!such!as!CFP!and!a!“acceptor”!fluorophore!such!as!YFP.!The!FRET!efficiency!that!occurs!between!the!two!proteins!is!calculated!by!comparing!the!CFP!and!YFP!intensities!before!and!after!destroying!the!acceptor!fluorophore!via!photobleaching.!If!FRET!is!occurring!between!the!two!proteins!there!should!be!an!increase!in!CFP!intensity!and!a!decrease!in!YFP!intensity!after!photobleaching.!Adapted!from!Yeon!Lee!(Wellesley!College,!NEURO!’15).!/

/

! 19!

Material/and/Methods:/

HEK!293!Cell!Culture,!Transfection,!and!Fixation:!

Human!embryonic!kidney!(HEK)!293!cells!(ATCC)!were!cultured!at!37°C!in!EMEM!

media!(ATCC)!with!10%!FBS!and!1%!penOstrep.!FRET!samples!were!prepared!by!plating!

HEK!293!cells!on!a!glass!coverslip!in!a!6Owell!plate!at!a!density!of!100,000!cells/coverslip.!

24!hours!after!plating,!cells!were!transfected!with!the!appropriate!cDNA!plasmid(s)!using!

XOtremeGENE!9!transfection!reagent!(Roche)!at!a!ratio!of!2!μl!XOtremeGENE!9!:!1!μg!DNA.!

Equal!amounts!of!DNA!(1ug!each)!were!used!for!FRET!pairs!that!required!coOtransfection!

of!two!different!plasmids.!Plasmids!and!XOtremeGene!9!were!diluted!in!EMEM!media!and!

allowed!to!incubate!for!30!minutes!before!being!added!dropwise!to!the!6Owell!plates!

containing!cells.!24!hours!postOtransfection,!the!media!was!refreshed.!Transfected!cells!

were!allowed!to!grow!for!an!additional!24!hours!(48!hours!total!postOtransfection)!and!

were!then!fixed!with!4%!paraformaldehyde!(Boston!BioProducts)!for!15!minutes.!After!

fixation,!the!cells!were!washed!in!PBS!(3x)!and!mounted!onto!slides!using!1O2!μl!of!

ProLongDiamond!(Life!Technologies).!!

To!address!the!question!of!whether!conjugating!the!donor!fluorophore!CFP!to!

KvLQT1!as!opposed!to!hERG,!affects!the!FRET!efficiency!between!the!two!proteins,!FRET!

samples!were!transfected!with!hERGOYFP!+!KvLQT1OCFP!(experimental!FRET!pair),!

KvLQT1OCFPOYFP!(positive!FRET!control),!and!hERGOCFP!(negative!FRET!control).!To!

address!the!question!of!whether!minK!affects!the!FRET!efficiency!between!hERG!and!

KvLQT1,!FRET!samples!were!transfected!with!hERGOCFP!+!KvLQT1OminK!OYFP!

(experimental!FRET!pair)!and!hERGOCFP!+!KvLQT1OYFP!(experimental!control).!!All!

fluorescent!proteins!used!were!monomeric,!except!KvLQT1OCFPOYFP!(positive!control).!All!

! 20!

proteins!were!–C!terminally!labeled!with!the!fluorescent!proteins,!and!in!the!case!of!

KvLQT1OminK!–YFP,!mink!was!directly!concatenated!with!KvLQT1!–N!terminally!labeled!

with!YFP.!

apFRET!Experimentation:!

FRET!samples!were!allowed!to!cure!for!at!least!24!hours!after!mounting!before!

apFRET!experiments!were!performed!using!confocal!microscopy.!CFP!and!YFP!

fluorophores!were!excited!with!458!nm!at!25%!transmission!or!514!nm!at!10%!

transmission,!respectively,!using!an!Argon!laser!at!20%!power!output.!The!fluorescence!

emission!of!the!CFP!or!YFP!fluorophore!was!detected!from!470O510!nm!and!580O650!nm,!

respectively.!512!x!512,!12Obit!images!of!cells!that!showed!expression!of!the!fluorescently!

labeled!CFP!or!YFP!protein!of!interest!were!obtained!using!an!63x!Plan!Apo,!NA=1.40!oil!

objective!lens!with!a!pinhole!of!1!Airy!Unit.!In!the!case!of!FRET!pairs!that!had!coexpression!

of!CFP!and!YFP!only!image!of!cells!that!qualitatively!appeared!to!have!similar!intensities!of!

expression!of!the!CFP!and!YFP!were!obtained!and!used!for!apFRET!experiments.!!

In!order!to!perform!apFRET!experiments,!a!circular!experimental!ROI!2.5!μm!in!

diameter!was!drawn!on!the!membrane!of!the!cell!so!that!half!of!the!area!of!the!circle!was!

within!the!cell!space!and!the!other!half!was!outside!of!the!cell!space,!!(Figure!10a,!10b).!The!

acceptor!YFP!fluorophores!within!the!ROI!were!then!photobleached!using!the!514!nm!

laser,!at!30%!transmission,!for!5!rapid!scans.!The!FRET!efficiency!was!then!calculated!by!

the!FRET!AB!Wizard!in!the!Leicia!LAS!AF!program!using!the!equation!FRETeff=(DpostO

Dpre)/Dpost!where!Dpost!is!the!fluorescence!intensity!of!the!donor!after!acceptor!

photobleaching,!and!Dpre!is!the!fluorescence!intensity!of!the!donor!before!acceptor!

photobleaching.!!

! 21!

After!bleaching!of!the!experimental!ROI,!an!analogous!control!ROI!was!drawn!on!the!

“preObleach”!image!of!each!cell!in!an!area!that!was!distinct!from!the!experimental!RO1!

(Figure!6A/B).!The!FRET!efficiency!was!calculated!in!this!control!ROI!in!which!bleaching!of!

the!acceptor!fluorophore!did!not!occur!by!the!FRET!AB!Wizard!program!using!same!FRETeff!

equation!above.!Figure!6!shows!representative!placements!of!the!experimental!and!control!

ROIs,!as!well!as!well!as!table!showing!the!mean!intensity!values!of!CFP!and!YFP!within!the!

ROIs!and!the!subsequently!calculated!FRET!efficiencies!for!the!positive!FRET!control!

KvLQT1OCFPOYFP.!!

!

! 22!

Figure/6./apFRET/results/for/the/positive/control/KvLQT1ICFPIYFP./A!2.5!μm!diameter!circular!bleach!region,!ROI!1,!was!drawn!on!the!plasma!membrane!of!Human!Embryonic!Kidney!(HEK)!Cells!expressing!KvLQT1!tagged!with!both!the!donor!fluorophore,!CFP,!and!acceptor!fluorophore,!YFP.!The!fluorescent!intensities!of!the!CFP!and!YFP!were!measured!in!this!region!prior!to!(A)!and!postO!(B)!YFP!bleaching!with!a!514nm!laser!at!20%!power!and!30%!transmission!for!5!scans.!For!each!cell!there!is!also!an!analogous!control!region,!ROI!2,!in!which!the!florescence!intensities!of!the!CFP!and!YFP!were!measured!in!the!absence!of!bleaching!with!the!514nm!laser.!The!preO!and!postObleach!intensity!values!of!CFP!and!YFP!and!the!FRET!efficiency!for!the!bleach!and!control!region!are!shown!in!C.!!/

! 23!

Results://!Optimization!of!apFRET!experimental!parameters:!!!

Prior!to!performing!apFRET!experiments!to!address!the!questions!of!whether!

interchanging!the!fluorophores!that!hERG!and!KvLQT1!are!attached,!or!whether!the!

concatenation!of!minK!to!KvLQT1!affects!the!FRET!efficiency!between!hERG!and!KvLQT1!it!

was!necessary!to!determine!the!parameters!used!by!the!FRET!AB!Wizard.!The!optimal!

transmission!percentages!of!the!458!nm!and!514!nm!lasers!used!to!excite!the!donor!(CFP)!

acceptor!(YFP)!respectively!were!previously!determined!to!be!25%!and!10%!respectively!

(Lee!’15).!Photobleaching!of!the!acceptor!(YFP)!fluorophore!was!optimally!achieved!by!

using!the!514!nm!laser!at!30%!transmission!for!5!rapid!scans!(Lee!‘15).!Lee!also!

determined!the!optimal!detection!ranges!for!the!donor!(CFP)!and!acceptor!(YFP)!to!reduce!

issues!of!excitation!crossOtalk!and!emission!bleedOthrough!between!the!donor!(CFP)!and!

acceptor!(YFP)!channels.!!

Excitation!crossOtalk!is!a!common!problem!in!fluorescence!microscopy!that!occurs!

when!the!wavelength!of!light!used!to!excite!one!fluorescent!marker,!such!as!YFP,!also!

excites!another!fluorescent!marker,!such!as!CFP!(Figure!7A).!Emission!bleedOthrough!is!a!

similarly!related!problem,!that!occurs!when!the!fluorescence!emission!of!a!fluorescent!

marker!is!detectable!in!a!channel!other!than!the!one!of!interest.!!Examples!of!emission!

bleedOthrough!are!shown!in!figure!7B!in!which!CFP!is!detected!in!the!YFP!channel.!!

! 24!

/Figure/7./Cartoon/representation/of/CFP/YFP/excitation/crossItalk/(A)/and/emission/bleedIthrough/(B)/within/cells/expressing/CFP./(A)!Excitation!crossOtalk!between!CFP!and!YFP!occurs!when!excitation!light!used!to!excite!YFP!also!excites!CFP!or!vice!versa.!(B)!Emission!bleedOthrough!between!CFP!and!YFP!occurs!when!CFP!emission!is!detectable!in!the!YFP!channel!or!vice!versa.!!!

When!imaging!a!sample!that!has!more!than!one!fluorescent!marker,!such!as!in!the!

case!of!apFRET!experiments,!it!is!important!to!avoid!issues!of!excitation!crossOtalk!and!

emission!bleedOthrough.!This!is!because!excitation!crossOtalk!and!emission!bleedOthrough!

make!it!impossible!to!determine!which!fluorescent!marker!the!detected!emission!

! 25!

originated!from.!Excitation!crossOtalk!and!emission!bleedOthrough!between!the!YFP!and!

CFP!fluorophores!is!a!problem!because!of!the!overlap!of!their!fluorescent!spectra!(Figure!

8A).!While!these!issues!cannot!be!completely!eliminated,!it!is!possible!to!choose!donor!

(CFP)!and!acceptor!(YFP)!detection!ranges!that!minimize!their!effects.!Furthermore,!it!is!

important!to!document!the!extent!to!which!excitation!crossOtalk!and!emission!bleedO

through!occurs,!as!these!are!errors!systemic!to!apFRET!experiments!that!have!relevancy!to!

the!interpretation!of!results.!!

Specifically!for!the!FRET!pair!CFP/YFP,!excitation!crossOtalk!of!YFP!by!the!CFP!458!

nm!laser!is!a!problem!due!to!the!fact!that!YFP!is!also!excited!by!458!nm!light!(Figure!8A).!In!

comparison,!excitation!crossOtalk!of!CFP!by!the!514!nm!YFP!laser!is!less!significant!because!

CFP!is!not!as!significantly!excited!by!514!nm!light!(Figure!8A).!Emission!bleedOthrough!is!

also!a!problem!for!CFP!and!YFP!due!to!their!overlapping!emission!spectra!(Figure!8B).!

BleedOthrough!into!the!YFP!channel!is!a!significant!issue!due!to!the!fact!that!a!portion!of!

CFP’s!emission!spectra!overlaps!with!the!entirety!of!YFP’s!emission!spectra,!from!roughly!

515!nm!onwards!(Figure!8B/C).!Thus,!it!is!likely!impossible!to!choose!YFP!detection!range!

in!which!CFP!emission!will!not!also!be!detected.!YFP!emission!bleedOthrough!into!the!CFP!

channel!is!less!of!a!problem!because!YFP’s!emission!spectra!does!not!overlap!with!the!

largest!and!peak!portion!of!CFP’s!emission!spectra!(450O500!nm)!(Figure!8B/C).!Thus,!it!is!

possible!to!choose!a!CFP!detection!range!in!which!YFP!does!not!emit.!In!summary,!in!

apFRET!experiments!that!employ!CFP!as!the!donor!and!YFP!as!the!acceptor,!excitation!

crossOtalk!and!emission!bleedOthrough!need!to!be!reduced!in!order!to!minimize!these!

artifacts!as!much!as!possible,!leading!to!more!accurate!measurement!of!the!preObleach!and!

! 26!

postObleach!donor!(CFP)!and!acceptor!(YFP)!fluorescent!intensities!used!to!calculate!the!

FRET!efficiency!(Figure!8B/C).!!

!

!Figure/8.!!Excitation/and/emission/spectra/of/CFP/and/YFP.!(A)!Excitation!spectra!of!CFP!(blue)!and!YFP!(yellow).!(B)!Emission!spectra!of!CFP!(blue)!and!YFP!(yellow).!(C)!Emission!spectra!of!CFP!(blue)!and!YFP!(yellow),!with!the!optimal!donor!(CFP)!detection!range!(470O510!nm)!and!acceptor!(YFP)!detection!range!(580O650!nm)!as!determined!by!Lee,!shown!in!grey.!(D)!Spectral!overlap!of!CFP!emission!spectra!(blue)!and!YFP!excitation!spectra!(yellow),!with!the!overlapping!area!is!shown!in!grey.!Image!adapted!from!Piston!et.!al,!2007.!!!

Lee!determined!that!the!optimal!detection!ranges!for!the!donor!(CFP)!and!acceptor!

(YFP)!were!470O510!nm!and!580O650!nm!respectively!(Figure!8C).!However,!reOalignment!

of!the!lasers!on!the!confocal!microscope!took!place!during!the!summer!of!2015!making!it!

necessary!to!reOoptimize!the!emission!detection!wavelength!ranges.!Consequently,!

performing!this!optimization!was!the!first!aim!of!my!thesis,!and!was!necessary!to!complete!

prior!to!performing!apFRET!experiments.!!!

!!!

! 27!

Acceptor!(YFP)!detection!range:!!

The!YFP!emission!spectra!ranges!from!500O650!nm,!with!a!peak!emission!at!~527!

nm!(Figure!8B).!In!order!to!determine!the!ideal!acceptor!(YFP)!detection!range,!three!

questions!were!considered:!(1)!What!wavelength!range!allows!for!maximal!detection!of!

YFP!fluorescence?,!(2)!What!wavelength!range!best!reduces!excitation!crossOtalk!of!YFP!by!

the!458!nm!CFP!laser?,!and!(3)!What!wavelength!range!best!reduces!CFP!emission!bleedO

through!into!the!YFP!channel?!In!choosing!potential!acceptor!(YFP)!ranges!to!test!I!sought!

to!test!ranges!that!balanced!the!amount!of!YFP!emission!and!CFP!emission,!such!that!I!

could!find!a!range!that!would!allow!for!a!strong!enough!detection!of!YFP,!while!limiting!the!

amount!of!CFP!emission!bleedOthrough!into!the!YFP!channel,!which!would!contribute!to!the!

acceptor!(YFP)!preObleach!and!postObleach!measurements.!This!was!a!difficult!task!because!

CFP!emission!spectra!significantly!overlapped!with!the!entirety!of!YFP’s!emission!spectra.!

Ultimately,!I!decided!to!test!the!wavelength!ranges!of!580O650!nm!and!590O650!nm!

because!both!of!these!acceptor!(YFP)!ranges!have!been!shown!successfully!allow!for!

detection!of!FRET!within!the!Darling!lab!(Lee,!2015).!!

In!order!to!determine!what!range!allowed!for!maximum!detection!of!YFP!

fluorescence,!I!measured!the!fluorescence!intensity!of!HEK!293!cells!expressing!KvLQT1O

YFP!that!were!excited!with!514!nm!light!at!10%!transmission.!Specifically,!the!fluorescence!

intensity!was!measured!within!circular!ROIs!(2.5!µm!in!diameter)!that!were!placed!either!

within!cells!that!were!fluorescing!(experimental!ROI)!or!within!the!background,!off!cellO

space!(control!ROI)!(Figure!9).!In!all!experiments!a!single!RO1!was!placed!within!each!cell.!

In!order!to!address!the!issue!of!YFP!excitation!crossOtalk!by!the!458!nm!CFP!laser,!I!

sought!to!determine!if!YFP!was!excited!by!the!CFP!laser.!In!order!to!do!this!the!fluorescence!

! 28!

intensity!was!measured!within!the!same!experimental!and!control!ROIs!that!were!

previously!drawn!on!the!HEK!293!cells!expressing!KvLQT1OYFP.!However,!for!this!

experiment!cells!were!excited!with!458!nm!at!25%!transmission!light!instead!of!514!nm!

light!at!10%!transmission.!In!total,!20!experimental!and!20!control!ROIs!were!drawn.!!

Finally,!in!order!to!address!CFP!emission!bleedOthrough!into!the!YFP!channel,!I!

sought!to!determine!if!CFP!fluorescence!was!visible!within!the!YFP!channels.!In!order!to!do!

this!HEK!293!cells!expressing!hERGOCFP!were!excited!with!458!nm!light!at!25%!

transmission,!and!the!fluorescence!intensity!was!measured!between!the!ranges!of!580O650!

nm!and!590O650!nm!within!analogous!experimental!and!control!ROIs.!In!total,!11!

experimental!and!11!control!ROIs!were!drawn.!A!scheme!outlining!the!experiments!

performed!in!order!to!determine!the!acceptor!(YFP)!detection!range!is!shown!in!figure!10.!!

!

! 29!

!Figure/9./Representative/image/of/experimental/(ROI/1IROI/4)/and/control/(ROI/4IROI/8)/ROIs/drawn/on/HEK/293/cells/expressing/KvLQT1IYFP/excited/by/514/nm/light/at/10%/transmission./!Each!ROI!was!2.5!µm!in!diameter!and!the!fluorescence!intensity!was!measured!at!various!wavelength!ranges!in!order!to!determine!the!optimal!acceptor!emission!wavelength!range!to!use!in!apFRET!experiments.!In!this!image!the!detection!wavelength!was!set!to!be!580O650!nm,!and!a!63x!Plan!Apo!oil!objective!with!a!NA!of!1.40!was!used.!!

! 30!

/Figure/10./Scheme/depicting/how/the/acceptor/(YFP)/detection/range/was/determined./(A)!To!determine!the!range!which!allowed!for!maximum!detection!of!YFP!fluorescence,!HEK!293!cells!expressing!KvLQT1OYFP!were!excited!with!514!nm!light!at!

! 31!

10%!transmission,!and!the!fluorescence!intensity!was!measured!between!the!wavelength!ranges!of!580O650!nm!and!590O650!nm!within!circular!control!and!experimental!ROIs.!(B)!To!determine!the!wavelength!range!that!best!reduced!excitation!crossOtalk,!HEK!293!cells!expressing!KvLQT1OYFP!were!excited!with!458!nm!light!at!25%!transmission,!and!the!fluorescence!intensity!was!measured!between!the!wavelength!ranges!of!580O650!nm!and!590O650!nm!within!experimental!and!control!ROIs.!(C)!To!determine!the!wavelength!range!that!best!reduced!emission!bleedOthrough,!HEK!293!cells!expressing!hERGOCFP!were!excited!with!458!nm!light!at!25%!transmission,!and!the!fluorescence!intensity!was!measured!between!the!ranges!of!580O650!nm!and!590O650!nm,!within!circular!control!and!experimental!ROIs.!!!

I!analyzed!the!results!of!the!previously!described!experiments!in!order!to!determine!

the!ideal!acceptor!(YFP)!detection!range.!Figure!11!shows!the!mean!fluorescence!intensity!

between!the!wavelength!ranges!of!580O650!nm!and!590O650!within!the!experimental!and!

control!ROIs!for!a!cell!expressing!KvLQT1OYFP!and!a!cell!expressing!hERGOCFP!

representative!of!the!populations!tested.!I!found!that!the!fluorescence!intensity!of!HEK!293!

cells!expressing!KvLQT1OYFP!excited!by!514!nm!light!at!10%!transmission!was!higher!

within!the!580O650!nm!range!compared!to!the!590O650!nm!range!(Figure!11A,!Figure!

12A/C).!This!indicates!that!the!580O650!nm!range!allows!for!a!stronger!detection!of!YFP!

fluorescence!compared!to!the!590O650!nm!range.!!

The!fluorescence!intensity!of!HEK!293!cells!expressing!KvLQT1OYFP!excited!by!458!

nm!light!at!25%!transmission!was!comparable!between!the!580O650!nm!and!590O650!nm!

ranges,!indicating!that!both!ranges!have!a!similar!level!of!excitation!crossOtalk!of!YFP!by!the!

458!nm!(CFP)!laser!(Figure!11B,!Figure!12B/D).!Additionally,!the!fluorescence!intensity!of!

KvLQT1OYFP!excited!by!458!nm!light!at!25%!transmission!was!lower!than!that!of!KvLQT1O

7FP!excited!by!514!nm!light!at!10%!transmission!within!both!the!experimental!and!control!

regions!(Figure!11A/B,!Figure!12).!

The!fluorescence!intensity!of!HEK!293!cells!expressing!hERGOCFP!excited!by!458!nm!

light!at!25%!transmission!was!comparable!for!both!the!580O650!nm!and!590O650!nm!

! 32!

ranges!tested!(Figure!11C,!Figure!13).!This!demonstrated!that!the!CFP!emission!bleedO

through!into!the!YFP!channel,!was!comparable!for!the!580O650!nm!and!590O650!nm!ranges.!

As!expected!the!CFP!intensity!within!the!580O650!nm!and!590O650!nm!ranges!was!much!

less!than!the!CFP!intensity!vissbile!within!the!CFP!channel!(Figure!13C)!Additionally,!the!

amount!of!bleedOthrough!into!the!580O650!nm!and!590O650!nm!channel.!!

/Figure/11./Mean/fluorescence/intensity/of/HEK/293/cells/expressing/KvLQT1IYFP/excited/by/either/514/nm/or/458/nm/light/and/HEK/293/cells/expressing/hERGICFP/exited/by/458/nm/light/between/the/wavelength/ranges/of/580I650/nm/and/590I650/nm./HEK!293!cells!expressing!KvLQT1OYFP!were!excited!with!514!nm!light!at!10%!transmission!(A)!or!458!nm!light!at!25%!transmission!(B)!and!the!fluorescence!intensity!was!measured!within!experimental!ROIs!placed!within!the!cell!(solid!lines)!and!control!ROIs!placed!within!the!background,!off!cell!space!(dashed!lines).!HEK!293!cells!expressing!hERGOCFP!were!excited!with!458!nm!light!at!10%!transmission!(C)!and!the!fluorescence!intensity!was!measured!within!experimental!ROIs!placed!within!the!cell!(solid!lines)!and!control!ROIs!placed!within!the!background,!off!cell!space!(dashed!lines).!!

! 33!

/Figure/12./HEK/293/cells/expressing/KvLQT1IYFP/were/not/significantly/excited/by/458/nm/light/and/showed/brighter/fluorescence/for/the/detection/wavelength/of/580I650/nm,/compared/to/590I650nm/when/excited/by/514/nm/light./HEK!293!cells!expressing!KvLQT1OYFP!were!excited/with!514!nm!light!at!10%!transmission!and!the!fluorescence!emission!was!detected!between!the!wavelength!ranges!of!580O650!nm!(A)!and!590O650!nm!(C).!In!order!to!address!the!issue!of!excitation!crossOtalk!of!YFP!by!the!CFP!laser!the!cells!were!also!excited!with!458!nm!light!at!25%!transmission!between!the!wavelength!ranges!of!580O650!nm!(B)!and!590O650!nm!(D).!Image!was!taken!using!63x!Plan!Apo!oil!objective!with!a!NA!of!1.40.!!!

! 34!

/Figure/13./Emission/bleedIthrough/of/HEK/293/cells/expressing/hERGICFP/within/the/YFP/channel/was/a/significant/issue/for/the/detection/wavelength/ranges/tested./HEK!293!cells!expressing!hERGOCFP!were!excited!with!458!nm!light!at!25%!transmission!and!the!fluorescence!intensity!was!measured!between!the!wavelength!ranges!of!580O610!nm!(A)!and!590O650!nm!(B).!The!intensity!of!!HEK!293!cells!expressing!hERGOCFP!excited!by!458!nm!light!within!the!CFP!channel!(470O510!nm)!is!shown!for!comparison!(C).!Image!was!taken!using!63x!Plan!Apo!oil!objective,!with!a!NA!of!1.40.!Scale!bars!represent!10!µm.!!!!

The!ideal!acceptor!(YFP)!detection!range!is!one!that!allows!for!a!strong!detection!

fluorescence!emission!of!YFP!that!is!excited!by!the!514!nm!light,!and!limits!the!detection!of!

fluorescence!emission!of!YFP!excited!by!458!nm!light!(excitation!crossOtalk).!Furthermore,!

the!ideal!acceptor!(YFP)!detection!range!should!limit!the!fluorescence!emission!of!CFP!

excited!by!the!458!nm!light!(emission!bleedOthrough).!The!issues!of!excitation!crossOtalk!of!

YFP!by!the!458!nm!(CFP)!laser!and!CFP!emission!bleedOthough!into!the!YFP!channel!were!

comparable!for!the!580O650!nm!and!590O650!nm!ranges!tested.!For!this!reason!I!

ultimately,!I!decided!that!the!ideal!acceptor!(YFP)!detection!range!was!580O650!nm!

because!this!range!it!yielded!stronger!detection!of!YFP!fluorescence!emission!compared!to!

the!590O650nm!range,!and!the!issues!of!excitation!crossOtalk!of!CFP!by!the!514!nm!(YFP)!

laser!and!CFP!emission!bleedOthrough!into!the!YFP!channel!were!not!any!worse!for!this!

range.!!

!!!!

! 35!

Donor!(CFP)!detection!range:!!

The!CFP!emission!spectra!ranges!from!460O650!nm!and!has!two!peaks,!one!at!~465!

nm!and!another!at!~510!nm!(Figure!8B).!In!order!to!determine!the!ideal!donor!(CFP)!

detection!range,!experiments!analogous!to!the!ones!used!to!identify!the!optimal!acceptor!

(YFP)!detection!range!were!performed.!I!considered!the!questions:!(1)!What!wavelength!

range!allows!for!maximal!detection!of!CFP!florescence?,!(2)!What!wavelength!range!best!

reduces!CFP!excitation!crossOtalk!from!the!514!nm!YFP!laser,!and!(3)!What!wavelength!

range!best!reduces!YFP!emission!bleedOthrough!into!the!CFP!channel?!I!chose!to!test!the!

wavelength!ranges!of!460O500!nm,!460O510!nm,!470O500!nm,!and!470O510!nm!because!

these!ranges!included!the!primary!CFP!emission!peak,!but!excluded!higher!wavelength!

ranges!where!YFP!emission!bleedOthrough!into!the!CFP!channel!is!likely!to!be!a!substantial!

artifact.!!

In!order!to!determine!which!range!allowed!for!maximum!detection!of!CFP!

fluorescence!I!measured!the!fluorescence!intensity!of!HEK!293!cells!expressing!hERGOCFP!

that!were!excited!with!458!nm!light!at!25%!transmission.!Specifically,!the!fluorescence!

intensity!was!measured!within!circular!ROIs!(2.5!µm!in!diameter)!that!were!placed!either!

within!cells!that!were!fluorescing!(experimental!ROI)!or!within!the!background,!off!cellO

space!(control!ROI)!as!demonstrated!in!Figure!14.!In!all!experiments!a!single!ROI!was!

placed!within!each!cell.!In!order!to!address!the!issue!of!excitation!crossOtalk,!I!sought!to!

determine!if!CFP!was!excited!by!the!YFP!laser.!In!order!to!do!this!the!fluorescence!intensity!

was!measured!within!the!same!experimental!and!control!ROIs!as!previously!drawn!HEK!!

293!cells!expressing!hERGOCFP.!!However,!in!this!case!the!cells!were!excited!with!514!nm!

! 36!

light!at!10%!transmission!instead!of!458nm!light!at!25%!transmission.!In!total!I!measured!

the!fluorescent!intensity!within!11!experimental!and!11!control!RO1s.!!

Finally,!in!order!to!address!the!issue!of!YFP!emission!bleedOthrough!into!the!CFP!

channel,!I!sought!to!determine!if!YFP!fluorescence!was!visible!within!the!CFP!channel.!HEK!

293!cells!expressing!KvLQT1OYFP!were!excited!with!514!nm!light!at!10%!transmission.!The!

fluorescence!intensity!was!measured!between!the!ranges!of!460O500!nm,!460O510!nm,!

470O500!nm,!and!470O510!nm!within!experimental!and!control!ROIs!that!were!analogous!

to!the!ones!previously!described.!In!total,!I!measured!the!fluorescence!intensity!within!20!

experimental!and!20!control!RO1s.!A!scheme!outlining!the!experiments!performed!in!order!

to!determine!the!donor!(CFP)!detection!range!is!shown!in!Figure!15.!!

!!!!

! 37!

/Figure/14./Representative/image/of/experimental/(ROI/1I/ROI/4)/and/control/(ROI/5IROI/8)/ROIs/drawn/on/HEK/cells/expressing/hERGICFP/excited/by/458/nm/light./!Each!ROI!was!2.5!µm!in!diameter!and!the!fluorescence!intensity!was!measured!at!various!wavelength!ranges!to!determine!the!optimal!donor!emission!wavelength!range!to!use!in!apFRET!experiments.!In!this!image!the!detection!wavelength!was!set!to!460O510!nm!and!a!63x!Plan!Apo!oil!objective!with!a!NA!of!1.40.!!!!

! 38!

!Figure/15./Scheme/depicting/how/the/donor/(CFP)/detection/range/was/determined./(A)!To!determine!the!range!which!allowed!for!maximum!detection!of!CFP!fluorescence,!HEK!293!cells!expressing!hERGOCFP!were!excited!with!458!nm!light!at!25%!transmission!

! 39!

and!the!fluorescence!intensity!was!measured!between!the!wavelength!ranges!of!460O500!nm,!460O510!nm,!470O500!nm,!470O510!nm!within!circular!experimental!and!control!ROIs.!(B)!In!order!to!determine!the!wavelength!range!that!reduced!excitation!crossOtalk,!HEK!293!cells!expressing!hERGOCFP!were!excited!with!514!nm!light!at!10%!transmission!and!the!fluorescence!intensity!was!measured!between!the!wavelength!ranges!of!460O500!nm,!460O510!nm,!470O500!nm,!and!470O510!nm!within!circular!experimental!and!control!ROIs.!(C)!In!order!to!determine!the!wavelength!range!that!reduced!emission!bleedOthrough,!HEK!293!cells!expressing!KvLQT1OYFP!were!excited!with!514!nm!light!at!10%!transmission!and!the!fluorescence!intensity!was!measured!between!the!ranges!of!460O500!nm,!460O510!nm,!470O500!nm,!and!470O510!nm,!within!circular!experimental!and!control!ROIs.!!!!

I!analyzed!the!results!of!the!previously!described!experiments!in!order!to!determine!

the!ideal!donor!(CFP)!detection!range.!Figure!16!shows!the!mean!fluorescence!intensity!

between!the!wavelength!ranges!of!460O500!nm,!460O510!nm,!470O500!nm,!and!470O510!nm!

for!a!cell!expressing!hERGOCFP!and!a!cell!expressing!KvLQT1OYFP!that!is!representative!of!

the!population!tested.!I!found!that!the!fluorescence!intensity!of!HEK!293!cells!expressing!

hERGOCFP!excited!by!458!nm!light!at!25%!transmission!was!comparable!for!the!460O510!

nm!and!470O510!nm!wavelength!ranges,!and!that!this!fluorescence!intensity!was!greater!

than!that!observed!for!the!460O500!nm!and!470O500!nm!ranges!(Figure!16A,!Figure!17).!!

This!result!indicated!that!the!460O510!nm!and!470O510!nm!wavelength!ranges!allowed!for!

a!stronger!detection!of!CFP!fluorescence!compared!to!the!460O500!nm!and!470O500!nm!

ranges.!!

The!fluorescence!intensity!of!HEK!293!cells!expressing!hERGOCFP!excited!by!514!nm!

light!at!10%!transmission!was!comparable!across!the!460O500!nm,!460O510!nm,!470O500!

nm,!and!470O510!nm!ranges,!and!the!fluorescence!intensity!was!lower,!within!the!

experimental!ROIs!compared!to!the!control!ROIs!for!all!the!ranges!tested!(Figure!16B,!

Figure!17).!Additionally,!fluorescence!intensity!of!hERGOCFP!excited!by!514!nm!light!at!

10%!transmission!was!lower!than!that!of!hERGOCFP!excited!by!458!nm!light!at!25%!

! 40!

transmission!within!both!the!experimental!and!control!regions!(Figure!16A/B,!Figure!17).!

This!results!indicates!that!as!expected!from!the!excitation!spectra!of!CFP,!excitation!CFP!

crossOtalk!from!the!514!nm!(YFP)!laser!was!not!a!substantial!problem.!!

The!fluorescence!intensity!of!HEK!293!cells!expressing!KvLQT1OYFP!excited!by!514!

nm!light!at!10!%!transmission!was!comparable!between!the!460O510!nm!and!470O510!nm!

ranges,!and!between!the!460O500!nm!and!470O500!nm!ranges!(Figure!16C,!Figure!18).!The!

fluorescence!intensity!associated!with!the!460O510!nm!and!470O510!nm!ranges!was!greater!

than!that!associated!with!the!460O500!nm!and!470O500!nm!ranges!(Figure!16C,!Figure!18).!

This!result!indicated!that!the!460O500!nm!and!470!nm!ranges!better!reduced!the!issue!of!

CFP!emission!bleedOthrough!into!the!YFP!channel!compared!to!the!460O510!nm!and!470O

500!nm!ranges.!!

!

!!!!!

! 41!

!Figure/16./Mean/fluorescence/intensity/of/HEK/293/cells/expressing/hERGICFP/excited/by/either/514/nm/or/458/nm/light/and/HEK/293/cells/expressing/KvLQT1IYFP/exited/by/458/nm/light/between/the/wavelength/ranges/of/460I500/nm,/460I510/nm,/470I500/nm,/470I510/nm./HEK!293!cells!expressing!hERGOCFP!were!excited!with!458!nm!light!at!25%!transmission!(A)!or!514!nm!light!at!10%!transmission!(B)!and!the!fluorescence!intensity!was!measured!within!experimental!ROIs!placed!within!the!cell!(solid!lines)!and!control!ROIs!placed!within!the!background,!off!cell!space!(dashed!lines).!HEK!293!cells!expressing!KvLQT1OYFP!were!excited!with!514!nm!light!at!25%!transmission!(C)!and!the!fluorescence!intensity!was!measured!within!experimental!ROIs!placed!within!the!cell!(solid!lines)!and!control!ROIs!placed!within!the!background,!off!cell!space!(dashed!lines)./!!!!

! 42!

/Figure/17./HEK/293/cells/expressing/hERGICFP/were/not/significantly/excited/by/514/nm/light/at/10%/transmission./HEK!cells!expressing!hERGOCFP!were!excited/with!either!458!nm!light!at!25%!transmission!or!514!nm!light!at!10%!transmission!and!the!CFP!emission!was!detected!between!the!wavelength!ranges!of!460O500!nm,!460O510!nm,!470O500!nm,!and!470O510!nm.!Image!was!taken!using!63x!Plan!Apo!oil!objective!with!a!NA!of!1.40.!Scale!bars!represent!10!µm.!!!!/

! 43!

/Figure/18./Emission/bleedIthrough/of/HEK/293/cells/expressing/KvLQT1IYFP/into/the/CFP/channel/was/minimal./HEK!cells!expressing!KvLQT1OYFP!were!excited!with!514!nm!light!at!10%!transmission!and!the!fluorescence!intensity!was!measured!between!the!wavelength!ranges!of!460O500!nm!(A),!460O510!nm!(B),!470O500!nm!(D),!and!470O510!nm!(E).!YFP!emission!of!KvLQT1OYFP!excited!with!514!nm!light!at!10%!transmission!between!the!wavelength!ranges!of!580O650!nm!(C)!and!590O650!nm!(F)!are!shown!for!comparison.!!!Image!was!taken!using!63x!Plan!Apo!oil!objective!with!a!NA!of!1.40.!Scale!bars!represent!10!µm.!/!!!

The!ideal!donor!(CFP)!detection!range!is!one!that!allows!for!a!strong!detection!

fluorescence!emission!from!CFP!excited!by!458!nm!light,!and!limits!the!detection!of!

fluorescence!emission!from!CFP!excited!by!514!nm!light!(excitation!crossOtalk).!

Furthermore,!the!ideal!donor!(CFP)!detection!range!is!one!that!limits!the!detection!of!

fluorescence!emission!from!YFP!excited!by!514!nm!light!(emission!bleedOthrough).!!Thus,!

there!was!a!conflict!in!choosing!the!best!detection!range;!the!460O510!nm!and!470O510!nm!

ranges!yielded!stronger!detection!of!fluorescence!emission!from!CFP!excited!by!458!nm!

! 44!

light,!but!the!460O500!nm!and!470O500!nm!showed!less!YFP!emission!bleedOthrough!into!

the!CFP!channel!(Figure!16).!I!ultimately!decided!that!having!a!wavelength!range!that!

maximized!the!detection!of!CFP!fluorescence!was!better!trade!than!having!slightly!reduced!

YFP!emission!bleedOthrough.!For!this!reason,!I!chose!to!focus!on!deciding!between!the!460O

510!nm!and!470O510!nm!donor!(CFP)!ranges!to!use!in!apFRET!experiments.!!

Considering!that!the!460O510!nm!and!470O510!nm!ranges!allowed!for!comparable!

detection!of!donor!(CFP)!fluorescence,!and!had!similar!amounts!of!acceptor!(YFP)!emission!

bleedOthrough,!I!performed!apFRET!experiments!on!KvLQT1OCFPOYFP,!the!FRET!positive!

control!sample,!in!order!to!determine!which!range!would!be!better!suited!for!apFRET!

experiments.!The!primary!goal!of!this!experiment!was!to!determine!if!the!two!ranges!

yielded!different!FRET!efficiencies,!and!if!so,!which!range!yielded!FRET!efficiencies!more!

similar!to!the!ones!previously!demonstrated!within!the!Darling!lab!and!reported!in!the!

literature!(Lee,!2015)!(OrganODarling!et!al.,!2013).!!

apFRET!experiments!were!performed!as!described!below,!and!the!580O650!nm!

acceptor!(YFP)!detection!range!was!used.!In!one!apFRET!experiment!I!used!the!donor!

(CFP)!detection!range!of!470O510!nm!and!in!another!I!used!the!donor!(CFP)!detection!

range!of!460O510!nm.!I!found!that!the!average!FRET!efficiency!for!the!470O510!nm!range!

was!27.6%!±!7.2%!(experimental!ROI,!n=24)!and!O1.3%!±!6.1%!(control!ROI,!n=24)!(Figure!

19A).!!I!found!that!the!average!FRET!efficiency!for!the!460O510!nm!range!was!24.9%!±!

6.6%!(experimental!ROI,!n=21)!and!O1.1%!±!5.5%!(control!ROI,!n=24)(Figure!19B).!The!

observed!FRET!efficiencies!for!the!460O510!nm!and!470O510!nm!ranges!were!similar!to!the!

ones!previously!demonstrated!within!the!Darling!lab!and!reported!in!the!literature,!and!

there!was!not!a!statistically!significant!difference!between!them!(p>0.01)!as!determined!by!

! 45!

a!Student’s!independent!tOtest.!For!this!reason,!I!decided!to!use!the!470O510!nm!range,!

because!this!was!the!same!range!Yeon!Joo!Lee!(’15)!of!the!Darling!lab!used!in!apFRET!

experiments!and!would!thus!allow!for!a!more!straightOforward!comparison!of!data!among!

lab!members.!!

!

/Figure/19./There/is/not/a/statistically/significant/difference/between/the/average/FRET/efficiencies/for/the/experimental/and/control/ROIs/for/KvLQT1ICFPIYFP/using/a/donor/(CFP)/detection/range/of/470I510/nm/(A)/and/460I510/nm/(B)./The!average!FRET!efficiencies!for!KvLQT1OCFPOYFP!associated!with!the!470O510!and!460O510!nm!donor!ranges!were!27.6%!±!7.2%!(experimental!ROI,!n=24)!and!O1.3%!±!6.1%!(control!ROI,!n=24)!and!24.9%!±!6.1%!(experimental!ROI,!n=21)!and!O1.1%!±!5.5%!(control!ROI,!n=24)!respectively.!!/!Control!apFRET!experiments:!!!

After!experimentally!determining!the!ideal!donor!(CFP)!and!acceptor!(YFP)!

detection!wavelength!ranges!to!be!470O510!nm!and!580O650!nm!respectively,!I!sought!to!

verify!that!these!wavelength!ranges!allowed!for!detection!of!FRET!via!apFRET!experiments.!

The!Darling!lab!has!previously!demonstrated!a!positive!FRET!efficiency!for!the!

! 46!

experimental!pair,!hERGOCFP!+KvLQT1OYFP,!and!the!positive!control,!KvLQT1OCFPOYFP,!

and!a!FRET!efficiency!that!is!not!statistically!different!from!the!control!region!for!the!

negative!control,!hERGOCFP,!via!apFRET.!I!sought!to!replicate!these!apFRET!experiments!to!

verify!that!the!selected!donor!(CFP)!and!acceptor!(YFP)!detection!wavelength!ranges!

allowed!for!detection!of!FRET.!!

For!all!apFRET!experiments,!cells!from!three!different!transfection!periods!imaged!

over!at!least!two!different!days,!were!pooled!into!one!sample!group.!This!accounted!for!

possible!variability!in!transfection!efficiency,!cell!passage!number,!and!fluctuations!in!the!

imaging!hardware!and!software.!As!a!result!of!noise!inherent!in!fluorescent!images,!for!

some!apFRET!experiments!the!donor!(CFP)!preObleach!intensity!was!lower!than!it!was!

postObleach.!This!is!resulted!in!a!negative!FRET!efficiency,!which!is!physically!impossible!to!

obtain.!In!these!instances,!the!Leica!imaging!software!reported!the!FRET!efficiency!as!0%.!

However,!for!the!purposes!of!data!analysis!I!calculated!the!negative!FRET!efficiency!based!

on!the!raw!data!readout!of!the!preObleach!and!postObleach!donor!(CFP)!intensities.!Ideally!

apFRET!experiments!were!performed!on!approximately!10O15!cells!per!slide,!and!each!

slide!was!imaged!in!a!single!sitting!to!ensure!that!cells!were!not!sampled!and!bleached!

more!than!once.!!

The!average!FRET!efficiencies!within!the!experimental!ROIs!for!hERGOCFP+KvLQT1O

YFP!(experimental!pair,!n=24),!KvLQT1OCFP+YFP!(positive!control,!n=24),!and!hERGOCFP!

(negative!control,!n=30)!were!O0.3%!±5!.6%,!27.6%!±!7.2%,!and!O0.7%!±!6.6%!respectively.!

The!average!FRET!efficiencies!within!the!control!ROIs!for!hERGOCFP!+KvLQT1OYFP!

(experimental!pair,!n=24),!KvLQT1OCFP+YFP!(positive!control,!n=24),!and!hERGOCFP!

! 47!

(negative!control,!n=30)!were!O0.4%!±!8.2%,!O1.3%!±!6.1%,!and!O1.7%!±!6.7%!respectively!

(Figure!19).!!!

There!was!not!a!statistically!significant!difference!between!the!control!and!

experimental!ROIs!for!hERGOCFP!+KvLQT1OYFP!(experimental!pair)!(p>0.01),!as!

determined!by!a!Student’s!independent!tOtest.!There!was!a!statistically!significant!

difference!between!the!average!FRET!efficiency!of!the!experimental!and!control!ROIs!for!

the!positive!control!KvLQT1OCFP+YFP!(p<0.001),!as!determined!by!a!Student’s!

independent!tOtest.!There!was!not!a!statistically!significant!difference!between!the!control!

and!experimental!ROIs!for!hERGOCFP!(negative!control)!(p>0.1),!as!determined!by!a!

Student’s!independent!tOtest.!!

!!

! 48!

Figure/19./There/is/a/statistically/significant/difference/between/the/average/FRET/efficiency/of/the/experimental/and/control/ROIs/only/for/the/positive/control/KvLQT1ICFPIYFP./Average!FRET!efficiencies!for!KvLQT1OCFPOYFP!(positive!control)!(a),!hERGOCFP!(negative!control)!(b),!and!hERGOCFP!+!KvlQT1OYFP!(experimental)!(c)!were!27.6%!±!7.2%!(experimental!ROI,!n=24)!and!O1.3%!±!6.1%!(control!ROI,!n=24),!O0.7%!±!6.6%!(experimental!ROI,!n=30)!and!O1.7%!±!6.7%!(control!ROI,!n=30),!and!0.3%!±5!.6%!(experimental!ROI,!n=24)!and!O0.4%!±!8.2%,!(control!ROI,!n=24),!respectively.!!!!!!!!!

! 49!

Experimental!apFRET!experiments:!!!

To!investigate!why!I!did!not!observe!a!positive!FRET!efficiency,!as!expected,!for!the!

experimental!pair!hERGOCFP!+!KvLQT1OYFP,!I!went!on!to!perform!apFRET!experiments!on!

the!experimental!pairs!hERGOYFP!+!KvLQT1OCFP!and!hERGOCFP!+!KvLQT1OminKOYFP!as!a!

means!of!troubleshooting.!These!apFRET!experiments!were!carried!out!in!an!analogous!

manner!to!the!control!apFRET!experiments!described!in!the!previous!section.!However,!the!

sample!size!for!these!experiments!was!smaller!and!apFRET!experiments!were!performed!

on!cells!pooled!from!two!different!transfection!periods,!as!opposed!to!three.!!

The!average!FRET!efficiency!in!experimental!ROIs!for!hERGOCFP!+!KvLQT1OYFP!

(n=15)!and!hERGOCFP!+!KvLQT1OminkOYFP!(n=20)!were!O0.1%!±!4.9%!and!O2.1%!±!6.1%,!

respectively.!The!average!FRET!efficiency!in!the!control!ROIs!for!hERGOCFP!+!KvLQT1OYFP!

(n=15)!and!hERGOCFP!+!KvLQT1OminKOYFP!(n=20)!was!O2.1%!±!5.4%!and!O1.1%!±!5.4%,!

respectively!(Figure!20).!There!was!not!a!statistically!significant!difference!between!the!

control!and!experimental!ROIs!for!hERGOCFP!+!KvLQT1OYFP!(p>0.1)!and!CFP+KvLQT1!+!

minkOYFP!(p>0.1),!as!determined!by!a!Student’s!independent!tOtest.!!

!

!

! 50!

!Figure/20./There/is/not/a/statistically/significant/difference/between/the/average/FRET/efficiencies/ for/ the/ experimental/ and/ control/ ROIs/ for/ hERGIYFP/ +/ KvLQT1ICFP/ or/hERGImCFP/+/KvLQT1IminKIYFP./The!average!FRET!efficiencies!for!hERGOYFP!+!KvLQT1OCFP!and!hERGOmCFP!+!KvLQT1OminKOYFP!were!O0.1%!±!4.9%!(experimental!ROI,!n=!15)!and!O2.1%!±!5.4%!(control!ROI,!n=15),!and!O2.1%!±!6.1%!(experimental!ROI,!n=20)!O1.1%!±!5.4%!(control!ROI,!n=20)!respectively.!!

!!!!!!!!!!!!!!!!!!!!!!!

! 51!

Discussion/and/Future/Directions://!

The!primary!goal!of!this!study!was!to!determine!whether!the!concatenation!of!minK!

to!KvLQT1!affects!the!FRET!efficiency!between!KvLQT1!and!hERG.!Concatenation!of!minK!

to!KvLQT1!is!necessary!in!order!for!proper!IKS!formation;!consequently,!in!order!to!perform!

patch!clamping!experiments,!which!offer!insights!into!the!function!of!hERG!and!KvLQT1,!it!

is!essential!that!minK!is!concatenated!to!KvLQT1.!Addressing!this!question!is!important!

because!performing!apFRET!experiments!within!the!same!experimental!situation!as!patch!

clamping!experiments!will!allow!for!functional!and!interaction!data,!collected!via!apFRET,!

to!be!compared.!This!is!of!interest!to!the!Darling!lab!because!the!complementation!of!

functional!data,!collected!via!patch!calming,!and!protein!interaction!data,!collected!via!

apFRET!experiments,!will!provide!a!clearer!picture!of!the!mechanism!of!the!interaction!

between!hERG!and!KvLQT1.!The!secondary!goal!of!this!study!was!to!address!whether!

interchanging!the!fluorophores!that!hERG!and!KvLQT1!are!concatenated!with!affects!the!

FRET!efficiency!between!the!two!proteins.!This!is!a!question!that!the!Darling!lab!has!been!

asked!to!resolve!by!multiple!grant!reviewers,!and!is!worth!answering!for!the!sake!of!

scientific!rigor.!!!

Prior!to!performing!apFRET!experiments!it!was!necessary!to!optimize!the!ideal!

donor!(CFP)!and!acceptor!(YFP)!wavelength!detection!ranges!to!be!used!in!apFRET!

experiments,!in!order!to!reduce!issues!of!excitation!crossOtalk!and!emission!bleedOthrough.!

While!it!is!not!possible!to!completely!eliminate!the!effects!of!excitation!crossOtalk!and!

emission!bleedOthrough,!it!is!possible!to!select!donor!(CFP)!and!acceptor!(YFP)!detection!

ranges!that!attenuate!their!effects.!Furthermore,!it!is!important!to!determine!the!extent!to!

! 52!

which!excitation!crossOtalk!and!emission!bleedOthrough!occur,!as!these!are!systemic!errors!

within!apFRET!experiments.!!

Lee!(’15)!previously!determined!the!ideal!donor!(CFP)!and!acceptor!(YFP)!detection!

ranges!to!be!460O510!nm!and!580O650!nm!respectively.!However,!during!the!summer!of!

2015!the!lasers!on!the!confocal!microscope!were!realigned,!making!it!necessary!to!reO

optimize!the!settings.!I!performed!similar!experiments!to!the!ones!Lee!(’15)!carried!out!and!

ultimately!confirmed!the!ideal!donor!(CFP)!and!acceptor!(YFP)!detection!ranges!to!be!460O

510!nm!and!580O650!nm.!Considering!Lee!(’15)!and!I!found!the!same!ideal!detection!

wavelength!ranges!suggests!that!reOalignment!of!the!lasers!did!not!have!as!significant!of!an!

impact!on!issues!of!excitation!crossOtalk!and!emission!bleedOthrough!as!previously!

imagined.!!

After!determining!the!ideal!donor!(CFP)!and!acceptor!(YFP)!detection!ranges,!I!went!

on!to!verify!that!I!could!reproduce!similar!FRET!results!as!the!ones!previously!

demonstrated!in!the!Darling!lab!and!reported!in!the!literature!(Lee,!2015,!OrganODarling,!

2013).!I!observed!a!positive!FRET!efficiency!of!27.6%!±!7.2%!(n=24)!within!the!

experimental!ROI!for!the!positive!control!KvLQT1OCFPOYFP.!This!result!was!expected,!

because!within!KvLQT1OCFPOYFP!the!CFP!and!YFP!fluorophores!are!linked!together!via!a!

short!peptide!sequence,!and!thus!can!easily!interact.!Furthermore,!the!FRET!efficiency!I!

observed!was!similar!to!the!FRET!efficiency!demonstrated!by!Lee!(’15)!and!OrganODarling!

et!al.!(2013)!who!observed!an!average!FRET!efficiency!within!the!experimental!ROI!of!25.8!

±!7.7%!(n=30)!and!22.0!±!4.0%!(n=39)!respectively.!!

For!the!negative!control,!hERGOCFP,!I!expected!to!observe!a!FRET!efficiency!close!to!

zero!percent!because!no!acceptor!(YFP)!fluorophore!was!present!for!intermolecular!FRET!

! 53!

to!occur.!I!observed!a!FRET!efficiency!of!O0.3%!±5!.6%!(n=30)!within!the!experimental!ROI.!

The!FRET!efficiency!I!observed!was!similar!to!that!that!demonstrated!by!Lee!(’15),!who!

observed!an!average!FRET!efficiency!within!the!experimental!ROI!of!2.5!±!3.7%.!OrganO

Darling!et!al.!(2013)!reported!an!average!FRET!efficiency!within!experimental!ROIs!for!

hERGOCFP!as!6.4!±!5.6%!(n=49),!which!was!higher!than!expected,!and!likely!a!result!of!

sensitivities!in!the!confocal!microscope!using!during!experimentation.!!!

I!observed!a!FRET!efficiency!of!O0.7%!±!6.6%!(experimental!ROI,!n=24),!which!was!

not!significantly!different!from!the!control!ROI,!which!had!a!FRET!efficiency!of!O0.4%!±!

8.2%!(n=24)!for!the!experimental!pair!KvLQT1OYFP+KvLQT1OCFP.!This!results!was!

unexpected!because!it!contradicted!the!results!of!previous!apFRET!performed!by!Lee!(’15)!

and!Organ!Darling!et!al.!(2013),!who!observed!intermolecular!FRET!between!the!

fluorophores!within!the!experimental!pair!KvLQT1OYFP+KvLQT1OCFP,!and!reported!the!

average!FRET!efficiencies!of!10.3!±!3.4%!(experimental!ROI,!n=30)!and!12.0!±!5.2%!

(experimental!ROI,!n=39)!respectively.!!Additionally,!this!result!contradicted!data!from!

biochemical!assays!that!demonstrate!an!interaction!between!hERG!and!KvLQT1.!For!

example,!Ren!et!al.!(2010)!showed!that!hERG!and!KvLQT1!physically!interact!via!coO

immunoprecipitation!assays,!and!this!finding!was!supported!by!Ehrlich!et!al.!(2004)!who!

showed!that!hERG!and!KvLQT1!coOimmunioprecipitated!in!cultured!CHO!cells!as!well!as!in!

native!cardiac!tissue!(Ren!et!al.,!2010,!Ehrlich!et!al.,!2004).!Given!the!body!of!evidence!for!

the!interaction!of!hERG!and!KvLQT1,!my!observation!that!the!FRET!efficiency!for!hERGO

CFP+KvLQT1OYFP!is!close!to!zero!percent,!suggests!that!the!apFRET!method!needs!further!

optimization!within!the!Darling!lab!before!it!can!be!used!to!study!the!mechanisms!of!

interaction!between!hERG!and!KvLQT1.!!

! 54!

It!is!especially!perplexing!that!I!could!not!reproduce!the!results!of!Lee!(’15)!who!

observed!intermolecular!FRET!between!the!fluorophores!in!the!experimental!pair!hERGO

CFP+KvLQT1OYFP,!considering!we!used!the!same!experimental!parameters!and!the!same!

confocal!microscope.!Factors!that!may!have!potentially!influenced!our!differing!results!are!

1)!variation!in!our!method!of!data!analysis!or!2)!selection!bias!in!the!cells!we!chose!to!

experiment!on!and!3)!selection!of!cells!with!hERG!or!KvLQT1!channels!were!not!fully!

functional!or!improperly!trafficked!within!the!cells.!!!

!Lee!(’15)!and!I!varied!in!our!method!of!data!analysis!in!how!we!dealt!with!negative!

FRET!efficiency!values.!While!a!negative!FRET!efficiency!has!no!physical!relevance!because!

it!is!impossible!for!the!postObleach!donor!(CFP)!intensity!to!be!greater!than!the!preObleach!

intensity,!negative!FRET!efficiencies!were!observed!during!experiments.!These!negative!

FRET!efficiencies!were!likely!a!result!of!noise!inherent!in!the!fluorescent!images.!The!Leica!

imaging!software!reports!negative!FRET!efficiencies!as!zero!percent!by!default,!and!in!

these!situations!I!reOcalculated!the!FRET!efficiencies!using!the!raw!data!readout!of!the!preO

bleach!and!postObleach!donor!(CFP)!intensities!and!used!the!negative!FRET!efficiencies!for!

data!analysis.!This!varied!from!Lee!(’15)!who!used!the!zero!percent!readout,!as!opposed!to!

the!actual!negative!FRET!efficiencies,!for!data!analysis.!!This!would!cause!Lee!(’15)!FRET!

efficiencies!to!be!artificially!higher!than!mine,!and!I!hypothesized!that!this!difference!in!

methodology!may!account!for!why!Lee!(‘15)!observed!a!statistically!significant!difference!

between!the!experimental!and!control!ROIs!for!the!hERGOCFP+KvLQT1OYFP,!and!I!did!not.!!

To!test!this!hypothesis!I!reanalyzed!my!data,!this!time!reporting!all!previously!

calculated!negative!FRET!efficiencies!as!zero!percent.!I!did!not!observe!a!difference!in!p–

values!for!the!negative!control!hERGOCFP!(p!=!0.53)!or!for!the!positive!control!KvLQT1O

! 55!

CFPOYFP!(p<0.0001)!when!I!reported!FRET!efficiencies!as!being!zero!percent!instead!of!

negative!values.!However,!for!the!experimental!pair!KvLQT1OYFP+hERGOCFP,!substituting!

zero!for!my!negative!FRET!efficiencies!in!the!analysis!lowered!the!pOvalue!from!p!=!0.29!to!

p!=!0.094.!Although!this!smaller!pOvalue!is!still!statistically!insignificant!at!the!95%!

confidence!level,!a!pOvalue!reduction!of!nearly!50!percent,!indicates!the!choice!of!!using!the!

actual!negative!efficiency!value!instead!of!identifying!the!negative!observation!as!zero!

percent!efficiency!does!change!the!magnitude!of!the!pOvalue!enough!to!potentially!impact!

the!interpretation!of!the!experimental!observations.!This!result!does!not!directly!answer!

the!question!of!whether!Lee!(’15)!would!have!observed!a!statistically!significant!difference!

between!the!experimental!and!control!ROIs!had!she!used!the!negative!FRET!efficiencies!in!

data!analysis,!and!it!would!be!worthwhile!to!reanalyze!her!data!specifically!to!address!this!

question.!!

Another!factor!to!consider!is!whether!biases!in!the!selection!of!cells!for!apFRET!

experiments!has!a!significant!impact!on!the!FRET!efficiencies.!There!is!a!large!degree!of!

variability!in!the!intensity!of!the!fluorescent!signal!of!transfected!cells,!with!some!cells!

being!much!brighter!than!others.!For!this!reason,!it!would!be!worthwhile!to!consider!

whether!cells!that!have!a!higher!level!of!fluorescent!protein!expression!yield!different!

FRET!efficiencies!than!cells!with!a!lower!fluorescent!protein!expression.!If!there!is!a!

difference,!then!possibly!selection!biases!between!the!cells!that!I!chose!to!include!in!my!

apFRET!experiments!and!those!that!Lee!(’15)!chose!to!include!could!lead!us!to!a!different!

result!for!the!experimental!pair!KvLQT1OYFP+hERGOCFP,!and!possibly!account!for!why!I!

did!not!observe!intermolecular!FRET.!!

! 56!

Yet!another!possibility!that!could!account!for!the!fact!that!I!did!not!observed!

intermolecular!FRET!for!the!experimental!pair!hERGOCFP+KvLQT1OYFP!is!if!the!hERG!or!

KvLQT1!channels!were!not!fully!functional!or!properly!trafficked!within!the!cells.!This!

could!result!from!either!damaged!plasmid!DNA!used!during!transfection,!or,!from!subO

optimal!cell!culture.!The!former!possibility!is!unlikely,!however,!as!I!observed!a!FRET!

efficiency!close!to!zero!percent!for!the!hERGOYFP+!KvLQT1OCFP!pair,!which!used!entirely!

different!plasmid!DNA!for!transfection,!and!the!hERGOCFP+KvLQT1OminKOYFP!pair,!which!

used!as!different!acceptor!(YFP)!plasmid!for!transfection.!!

If!hERG!and!KvLQT1!channels!were!not!properly!trafficked!or!fully!functional!such!

that!they!did!not!interact!as!they!would!in,vivo,!a!positive!FRET!efficiency!would!be!

observed!for!the!positive!control!KvLQT1OCFPOYFP.!Within!KvLQT1OCFPOYFP,!the!CFP!and!

YFP!fluorophore!are!both!directly!conjugated!to!the!KvLQT1!via!a!short!intervening!

sequence;!thus,!even!if!KvLQT1!was!not!fully!functional!or!was!missO!trafficked,!

intermolecular!FRET!between!the!two!fluorophores!would!still!occur.!Furthermore,!one!

would!still!observe!a!FRET!efficiency!close!to!zero!percent!for!the!negative!control,!hERGO

CFP,!because!regardless!of!the!functionality!or!location!of!hERG,!no!acceptor!(YFP)!

fluorophore!is!present!for!intermolecular!FRET!to!occur.!However,!for!intermolecular!FRET!

to!occur!between!CFP!and!YFP!in!the!cases!of!the!experimental!pairs!hERGOCFP+KvLQT1O

YFP,!hERGOYFP+KvLQTOCFP,!or!hERGOCFP+KvLQT1OminKOYFP,!it!is!necessary!for!KvLQT1!

and!hERG!channels!to!come!together,!which!would!require!functionality!and!proper!

translocation!of!the!channels.!CoOimmunoprecipitation!assays!in!combination!with!

biotinylation!assays,!which!address!whether!the!proteins!of!interest!are!properly!trafficked!

to!the!cell!membrane,!could!shed!light!on!whether!the!channels!are!properly!trafficked!and!

! 57!

functional.!If!hERG!and!KVLQT1!channels!were!confirmed!to!interact!via!coO

immunoprecipitation!assays,!it!would!indicate!that!the!apFRET!needed!further!optimizing!

to!detect!the!interaction!of!hERG!and!KvLQT1.!!!

An!additional!consideration!currently!being!addressed!is!whether!the!mounting!

media!used!to!prepare!slides!for!apFRET!experiments!reduces!the!sensitivity!of!detection!

of!intermolecular!FRET.!For!all!apFRET!experiments!I!used!ProLongDiamond!(Life!

Technologies)!mounting!media,!which!is!specifically!designed!to!reduce!photoObleaching!of!

florescent!proteins.!While!maintaining!a!robust!fluorescent!signal!is!generally!desirable!in!

most!fluorescent!imaging!contexts,!this!could!be!problematic!in!apFRET!experiments!that!

rely!on!successfully!bleaching!the!acceptor!(YFP)!fluorophore.!To!test!the!hypothesis!that!

ProLongDiamond!mounting!media!reduces!the!sensitivity!of!FRET!detection,!we!will!

compare!the!FRET!efficiency!and!bleach!depth!(percent!of!YFP!bleached!out)!of!KVLQT1O

CFPOYFP!using!slides!prepared!with!ProLongDiamond!mounting!media!and!ProLongGold!

(Life!Technologies)!mounting!media,!which!is!designed!to!reduce!photoObleaching!of!

fluorescent!labels!other!than!proteins.!If!it!is!shown!that!ProLongDiamond!reduces!the!

sensitivity!of!FRET!detection,!this!could!in!part!explain!why!I!did!not!observe!

intermolecular!FRET!for!the!experimental!pair!KvLQT1OYFP+hERGOCFP.!!

The!fact!that!I!did!not!observe!a!positive!FRET!efficiency!for!the!experimental!pair!

KvLQT1OYFP+hERGOCFP!is!an!unexpected!result!based!on!previously!published!findings!

and!onOgoing!work!within!the!Darling!lab.!Specifically,!the!outcome!of!Lee!(’15)!and!I!

finding!differing!FRET!efficiencies!for!this!pair!suggests!that!the!apFRET!method!needs!

further!optimization!within!the!Darling!lab.!Such!optimization!is!necessary!to!address!the!

! 58!

specific!aims!of!my!thesis,!as!well!as!to!more!broadly!investigate!the!mechanisms!of!

interaction!between!hERG!and!KvLQT1.!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

! 59!

References://!

1.! Brunner!M,!Peng!X,!Liu!GX,!Ren!XQ,!Ziv!O,!Choi!BR,!Mathur!R,!Hajjiri!M,!Odening!KE,!Steinberg!E,!Folco!EJ,!Pringa!E,!Centracchio!J,!Macharzina!RR,!Donahay!T,!Schofield!L,!Rana!N,!Kirk!M,!Mitchell!GF,!Poppas!A,!Zehender!M,!Koren!G.!Mechanisms/of/cardiac/arrhythmias/and/sudden/cardiac/death/in/transgenic/rabbits/with/long/QT/syndrome.!J,Clin,Invest,2008!118:!2246O2259.!!

2.! Curran!ME,!Splawski!I,!Timothy!KW,!Vincent!GM,!Green!ED,!Keating!MT:!A/Molecular/Basis/for/Cardiac/Arrhythmia:/HERG/Mutations/Cause/Long/QT/Syndrome.!Cell,1995,!80/795O803.!

3.! Ehrlich!JR,!Pourrier!M,!Weerapura!M,!Ethier!N,!Marmabachi!AM,!Hebert!TE,!Nattel!S.!KvLQT1/modulates/the/distribution/and/biophysical/properties/of/HERG./A/novel/alphaI/subunit/interaction/between/delayed/rectifier/currents//J,Biol,Chem,2004!279:1233O1241!!

4.! Grant!AO:!Cardiac/ion/channels.!Circ,Arrhythm,Electrophysiol,2009,!2(2):185O194.!5.! H!J,!Cabral!M,!Lee!A,!Cohen!SL,!Chait!BT,!Li!M,!Mackinnon!R:!Crystal!Structure!and!

Functional/Analysis/of/the/HERG/Potassium/Channel/N/Terminus:/A/Eukaryotic/PAS/Domain.!Cell,1998,!95:649–655.!

6.! Jespersen!T,!Grunnet!M,!Olesen!SOP:!The/KCNQ1/Potassium/Channel:/From/Gene/to/Physiological/Function.!Physiology,2005!20/208O416.!

7.! Kim!E,!Darling!LEO:!Optimization/of/coIimmunoprecipitation/assays/to/determine/the/molecular/mechanism/modulating/direct/interactions/between/potassium/channel/αIsubunit/proteins/hERG/and/KvLQT1/2015!

8.! Lee!YJ:!Determining/the/Molecular/Mechanism.!2015!9.! OrganODarling!LEO,!Vernon!AN,!Giovanniello!JR,!Lu!Y,!Moshal!K,!Roder!K,!Li!W,!

Koren!G.!Interactions/between/hERG/and/KCNQ1/αIsubunits/are/mediated/by/their/COOH/termini/and/modulated/by/cAMP.!Am,J,Physiol,Heart,Circ,Physiol,2013!304:!H589O!h599.!!

10.!Piston!DW,!Kremers!GJ:!Fluorescent/protein/FRET:/the/good,/the/bad/and/the/ugly.!Trends,Biochem,Sci,2007,!32(9):407O414.!

11.!Pusch!M:!Increase/of/the/singleIchannel/conductance/of/KvLQT1/potassium/channels/induced/by/the/association/with/minK.!Eur,J,Physiol,1998(437):172–174.!

12.!Ren!XQ,!Liu!GX,!OrganODarling!LE,!Zheng!R,!Roder!K,!Jindal!HK,!Centracchio!J,!McDonald!TV,!Koren!G.!Pore/mutants/of/HERG/and/KvLQT1/downregulate/the/reciprocal/currents/in/stable/cell/lines.!Am,J,Physiol,Heart,Circ,Physiol,2010,!299:!H1525O!H1534!

13.!Sesti!F,!Goldstein!S:!SingleIChannel/Characteristics/of/WildIType/IKs/Channels/and/Channels/formed/with/Two/MinK/Mutants/that/Cause/Long/QT/Syndrome.!J,Gen,Physiol,1998,!112:651–663.!

14.!Sherwood,!L.!Human/Physiology.!BroksCole.!2012,!8!(13).!�!15.!Splawski!I,!TristaniOFirouzi!M,!Lehmann!MH,!Sanguinetti!MC,!Keating!MT:!

Mutantions/in/the/hminK/gene/causes/long/QT/Syndrome/and/supresses/IKs/function/Nature,1997,!17:338O340.!

! 60!

16.!Vandenberg!JI,!Perry!MD,!Perrin!MJ,!Mann!SA,!Ke!Y,!Hill!AP:!hERG/K+/Channels:/Structure,/Function,/and/Clinical/Significance.!Physiological,Reviews,2012,!92(3):1393O1478.!

17.!Warmke!J,!Ganetzky!B:!A/family/of/potassium/channel/genes/related/to/eag/in/Drophila/and/mammals.!Proceedings,of,the,National,Academy,of,Science,1994,!91:3438O3442.!

18.!Yang!WOP,!Levesque!PC,!Little!WA,!Conder!ML,!Shalaby!FY,!Blanar!MA:!KvLQT1,/a/voltageIgated/potassium/channel/responsible/for/human/cardiac/arrhythmias.!Proceedings,of,the,National,Academy,of,Science,1997,!94:4017–4021.!!!

!