light gated ion channels
TRANSCRIPT
LIGHT GATED ION CHANNELS AND THEIR APPLICATIONS
BRIJESH CHANDRAKAR31st August, 2013
Introduction
• Ion channels are gated by a variety of stimuli.• Ligands, voltage, membrane tension,
temperature, light etc.• Channels can be altered, augmented to
develop channels with completely new functional properties.
Light gated ion channels
• Definition- A pore whose conductance reversibly changes in response to irradiation with UV or visible light.• Advantages- Optical manipulation offers a high degree of
spatial and temporal control. Such channels would allow entirely new control mechanisms for the exploration and manipulation of biological systems. When applied to nervous systems, such an approach could even result in the development of “artificial senses”.
Overview
• How chemical modifications are employed on ion channels to make them light sensitive?• Existing examples based on the type of
photoswitch employed.• Recent Development• Applications, possible research
prospectives.
Contents• Artificial • Nicotinic Acetlycholine Receptor (nAChR, agonist azobenzene)• Gramicidin A• Voltage gated K+ channels• Ionotropic Glutamate Receptor• Mechanosensitive channel
• Natural• Channelrhodopsin
Photochemistry• Photoswitches: control gating by changing local electric field,
tethering components, moving blockers or agonists or inducing conformational changes deep inside protein to open a conductive pathway.
• Photoswitching must occur on time scale of protein and cellular function using light intensities that are not harmful to cells.
off condition (stable) on condition (less stable) Retinal, Natural Photoswitch
Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:2
Algorithm for making ion channels light sensitive
Modify receptor to make it light sensitiveMutation in native receptor (inducting photoswitches) Functioning in biological conditions
Identify receptor regulating activitySite Type of receptor (stimuli)
Identify ion channelProduction Structure
Different artificially light gated ion channels
Nicotinic Acetylcholine Receptor (nAChR) A: Normal functioning native acetyl choline receptor
B: Light gated nAChR Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:3
trans-Bis-Q act as agonist : respond to light and activates nAChR.
trans Bis-Q trans Q-Br (nAChR inactive) (nAChR active)
Photoisomerization between the two states usingmillisecond light flashes produced rapid changes in theconcentration of trans isomer, resulting in reversible inductionof inward currents.
Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:3
Gramicidin A• Pentadecapeptide (antibiotic) derived from Becillus Brevis.
Native function of gamicidin A. Two monomers dimerize in a cell membrane to form a functional cation channel.
Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:4
Light gated Gramicidin ATwo monomers are linked by azobenzene that induces dimerization in cis form.
Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:4
Inactive (trans benzeneacetic acid) Active (cis benzeneacetic acid)
Voltage gated potassium channel• Have pore blocking tetraethyl ammonium derivative.• Connected to peptidic tether whose change in length
regulates opening/ blocking of pores.• Peptidic tether replaced by para-azodianiline which
changes length upon photochemical isomerization. • Such channels: SPARK (Synthetic Photoisomerizable
Azobenzene Regulated K+ channels)• SPARK mutated to minimize the effects of endogenous
gating mechanisms.
Light gated potassium ion channelsA. Native potassium ion channel.
B. Light gated ion channel Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:5
Ionotropic Glutamate Receptor
• In iGluR6, kinate receptors (iGluR5-6) possess an exit tunnel connecting the binding pocket and extracellular surface.• iGluR6 mutated • Cysteine at amino acid position 439 (L493C).• It covalently binds to cis-MAG: maleimide (M), an
azobenzene linker (A), and a glutamate headgroup (G).• Isomerization to the trans form of the azobenzene led to
a significant reduction in current (inactivation of the channel).
Light gated iGluRA. Native iGluR
B. Light gated iGluR (cis isomer of azobenzene activates channel) Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:7
Mechanosensitive Channel
• Upon the onset of hypo-osmotic shock, these channel gates response to changes in membrane tension.• Possess a hydrophobic gate within the pore that
effectively locks the channel in a closed state in the absence of membrane tension.• Mutation to polar or charged residues at amino
acid Gly22 significantly weakens this hydrophobic interaction, providing access to an expanded sub conducting state.
MscLA. native function of MscL. Membrane tension disrupts a hydrophobic gate to open the 30-40 Å
pore.B. light-gated MscL. A relatively nonpolar spiropyran is conjugated to the pore lumen such that photoconversion to the more polar merocyanine form weakens the hydrophobic gate to induce
pore opening. Biochemistry Volume 45, Number 51, 2006, American Chemical Society, page:9
Active (UV)Inactive (visible)
• Source- Green alga Chlamydomonas reinhardtii.• Maximally excited by 470nm light.• Excited by light.• ChR opens.• Depolarizes membrane.• Ion selectivity- Cation permeant. Non-selective towards H+, K+,
Na+ and Ca+2.• The covalently linked all-trans retinal chromophore of the ChRs
absorbs in the visible range.• Upon absorption of a photon, all-trans-retinal probably
isomerizes to 13-cis-retinal.
Channelrhodopsin (ChR2)
Optogenetic techniques
Recent Development• Cardiac Optogenetics- optical interrogation and control of
biological function with high specificity and high spatiotemporal resolution. Mammalian cells and tissues can be sensitized to respond to light by a relatively simple and well tolerated genetic modification using microbial opsins (light-gated ion channels and pumps). These can achieve fast and specific excitatory or inhibitory response, offering distinct advantages over traditional pharmacological or electrical means of perturbation.Further reading- http://ajpheart.physiology.org/content/early/2013/02/25/ajpheart.00432.2012
• Channelrhodopsins : visual regeneration and neural activation by a light switch- It has been shown that integration of ChR2 into various tissues of Mice can activate neural circuits, control heart Muscle contractions, and even restore breathing after spinal cord injury.Further reading- http://www.sciencedirect.com/science/article/pii/S1871678413000599
Applications and further researchLight gated ion channels• Tools for controlling the function of excitable cells or artificial
membrane vesicles.• It offers the opportunity to govern the electrical excitability of cells
and other membrane structures with a high degree of temporal and spatial control. Light gated nAChR could be of use in studying nicotine addiction and Alzheimer’s disease to a greater accuracy.
• Useful for restoring function downstream of neural damage, particularly in the retina, with hopes of creating some form of artificial vision.
• Can be incorporated into artificial lipid bilayers and liposomes, which could be utilized as artificial memory storage devices (gramicidin A) or as a way to optically induce targeted drug delivery.
• Biophysical investigations of the kinetics of ion channel opening may be facilitated by optical activation, which eliminates the limiting process of diffusion when applying ligands in solution.
• Features of the chromophores could be refined as well. Because most photoswitches are triggered by UV light, red-shifted photoswitches that operate fully in the visible light are needed. It will reduce toxicity associated with UV light.
References• Matthew R. Banghart, Matthew Volgraf, and Dirk Trauner,
(2006) ; Engineering light gated ion channels and their applications, Biochemistry, American Chemical Society Volume 45, Number 51.
• Emilia Entcheva (March, 2013), Cardiac Optogenetics, Articles in PresS. Am J Physiol Heart Circ Physiol.
• Natasha G, Aaron Tan, Yasmin Farhatnia, Jayakumar Rajdas, Michael R. Hamblin, Peng T. Khaw and Alexander M. Seifalian (June 2013), New Biotechnology, Volume-33, Number-5
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