Voltage-Gated Ca2+ Channels

History• Fatt & Katz (1953) discovered an exception to Na APs:– Na substitution made APs stronger!– TEA antagonism spread AP even more (why?)– procaine enhanced excitability!– thus, crustacean muscle doesn’t use Na+ for APs

• “Ca2+ spiking”• ECa even more positive than ENa

• Fatt & Ginsborg– AP requires Ca2+, Sr2+, or Ba2+

• Hagiwara & Naka (1964)– lowering [Ca]in (free) increases response in barnacle muscle– must be <0.1 mM for all-or-nothing response

[Ca]-sensitive Channels?

• some Ca channels are inactivated by [Ca]in > 0.1-1.0 mM• [Ca]in-sensitive K channels also exist, activated by ↑[Ca]in

• both mechanisms (decrease [Ca]in)would serve to generate the barnacle muscle cell experimental results

• No TTX-sensitive Na currents in arthropod muscle fibers, only in axons

• Hagiwara & Naka (1964) also measured 45Ca fluxes, to nail it definitively– 2-6 pmol of Ca2+ per mF of membrane C (only 0.5 needed for

cation to depolarize by 100 mV

Ubiquity of Ca Channels

• permeant divalent cation competition• divalent transition metal blockage (Ni2+, Cd2+, Co2+)• variety of subtypes, via characterization based on

– activation ranges– ion selectivity– sensitivity to blockage– kinetics

• functions– secretion– sarcomeric shortening (contraction)– exclusively for APs in myocytes of arthropods, nematodes,

tunicates, molluscs, and in vert. SmM, vert. CardM

Revelation Delayed

• Ca channels don’t occur on clampable membranes in high density with easy accessibility– nerve terminals, dendrites, infoldings of myocytes– nerve soma, confounded by other currents, e.g. K

• suction pipettes were developed for voltage clamp (precursor of gigaseal [109W] whole cell patch)

• gastropod ganglion cells were used• blockage of INa with TTX or substitution (externally)• blockage of IK with Cs+ or TEA substitution

(internally)

ICa• inward current• can be masked

by INa

• smaller magnitude (SA-normalized): ~40-50 mA/cm2 or 100x less than INa in vert. SkM, axons• slower than INa

why this shape?

Early Nomenclature• High-Voltage Activated (HVA)

– often lack rapid inactivation– require much larger depolarizations to activate than

• Low-Voltage Activated (LVA) Ca channels– usually have rapid Vm-dependent inactivation (measure peaks

for I-V curves)– activate in response to smaller depols

• Recall, classical interaction betweenVm and g• Ca channels have 2 major roles:

– electrical excitation– regulation

ICa Contributions

little INa

mostly ICa

high INa

ICa (HVA) maintains

depol

ICa(LVA), INa

ICa (HVA)

Ventricular Myocytes• Na+ influx depolarizes cell• Na+ Ch inactivation is not

accompanied by repolarization• plateau potential (~0 mV) is

maintained– K+ chs remain/close– Ca2+ chs open

• Ca2+ chs (Vm-gated) open slowly and remain open long (L-type)

• ICa2+ balances IK+

• repolarization occurs when gK+, gCa2+ return to original state

typical

long-lasting

atypical

Pacemaker Potentials• spontaneous, rhythmical

de/repolarizations• most prominent in SA node

Mechanisms:1. ↓PK+ due to previous AP2. opening of chs at polarized levels

(If): mostly Na+

3. T-type Ca2+ chs open

APs via L-type Ca2+ chs, repolarized after delay by IK+ chs

SA node captures other PMPs

typicalbut slow

Na+

Ca: Excitation & Messenger• neither the nervous,

skeletal muscle, cardiac muscle, nor endocrine systems are a purely electrical systems. They are electro-chemical/mechanical systems.• in addition to its role in

excitation, Ca is an important mediator in both mechanical force generation and cellular physiological responses

Why Ca as a 2nd Messenger?• free [Ca]in is deliberately

kept low (30-200 nM) by– Ca-ATPase pump (cell &

ER/SR membrane)– Na/Ca exchanger (cell

membrane)• EC gradient is always into

cytoplasm (either from ER/SR or from interstitial space)

• Unlike typical K or Na fluxes, Ca flux may have dramatic effect on [Ca], with huge effects on cellular processes

Biophysical Interests

• Contraction (muscle)• Secretion (neurotransmission, NMJ,

endocrine)• Gating (Ca, K channels)

Muscles• source of Ca is often SR (cardiac/neurons are more

complex, depend on both external and internal sources)• free cytosolic Ca binds to calmodulin, troponin, and other

molecules• this may result in activation (and inactivation) of classes

of enzymes, including kinases, that result in short-term and long-term physiological changes.

• in muscles, Ca binding uncovers myosin binding site on actin, and ATPase portion of actin-myosin complex is free to produce displacement (shortening), the physical underpinning of contraction

• Ca-calmodulin acts on cytoskeletal complexes to influence motility, migration

Synaptic Transmission• AP travels down axon to terminal• Depolarization opens Vm-gated Ca2+ channels (N-type)• Ca2+ flows into terminals, initiates exocytosis of vesicles

(i.e., vesicle fusion with presyn memb)• Prior to this, SNARE proteins interact in vesicle memb,

presynapt memb, and terminal cytoplasm• Once fusion occurs, vesicle contents (neurotrans, cotrans) is

released into synaptic cleft and diffuses to postsyn memb• Ions flow in as a result of NT binding to postsyn receptors.

Opening (1st mess) or shutting (2nd mess).• delay between arrival of AP & D in postsyn Vm ~0.2 ms

Synaptic Transmission1. AP invades axon terminals2. Vm-dep Ca2+ Chs open

3. [Ca2+]in rises

4. synaptic vesicles fuse to plasma memb

5. neurotransmitter released into synaptic cleft

6. NT binds to postsynaptic Rs, opening them

7. Cations or anions flow inThus, electrochemical

communication

Synaptic/NMJ Potentials• are inhibited by

Mg2+, which blocks Ca channels

• Squid giant synapse allowed for Ca imaging, voltage clamping, and injection of Ca directly.

• Can now image and patch onto vertebrate CNS synapses

• presynaptic [Ca] changes are rapid in onset

• buffered slowly• cumulative• Ca must be

pumped actively• at Ca chs near

synaptic vesicles: >100X greater rise and faster onset and decay

• unknown sensor molecules: several vesicle-associated proteins bind Ca

[Ca]in and Gating

• several Ca, K (0.1-0.9 mM), and Cl (0.2-0.8 mM) channels are sensitive to [Ca]in

• so are some non-specific cation channels (1-6 mM)

• NMDA channels conduct Ca2+ (large) and Na+ (smaller)

• all have binding sites for Ca2+ or calmodulin on intracellular surfaces

Why this shape?

Why this effect?

Note: this is spatial average!

Local [Ca]?

Why this effect?

What’s this about?KDR + K(Ca)

What’s the implication here?

why biggesteffect here?

IK

Bottom Line• Many [Ca]in-dependent processes are Vm-

dependent, since many Ca channels are Vm-dependent and Ca will enter from the extracellular space.

• These processes may or may not be Vm-sensitive irrespective of [Ca]in

Electrical Characterization

• Based on Vm-dependence & kinetics– HVA: large depol for act, slow kinetics, no rapid inact– LVA: small depol for act, faster kinetics, rapid Vm-dep

inact

How would you distinguish between them, experimentally, if they were both present in the

same cell?

Separation of HVA & LVA…by Vhold during

voltage-clamp

L- vs. T-type Nomenclature• based on unitary

conductance measurements

• L = long lasting, larger conductances (HVA)

• T= transient, tiny conductances (LVA)

• note: driving force on Ba2+ is less at more depolarized potentials, so currents are even more disproportional

Ca Channel Antagonists• clinical uses:– L-type blockers (cardiac and SmM) such as 1,4-dihydropyridines

(fig. 4.13):• D-600, nifedipine, nitrendipine, diltiazem: 20nM-50mM ½ blocking

– lipid-soluble drugs can block other channel types at high concentrations

• DHPs have complex effects– may ↑ (agonist; BAY K 8644) or ↓ (antagonist; nif., nit.) ICa

– same compound may serve as both, because they contain a mixture of 2 optical enantiomers (isomers)

• BAY K 8644 increases open time, probability of opening of L-type ICa

Vm-dependent Antagonism• binding of blockers is Vm-dependent: this is rule

Implication?• ~2000x more nitrendipine is needed to block L-type

channels at -80 mV than at -15 mV (see KD values)

inactivated“deinactivated”

Another Type of Ca Channel• “HVAish” channels have been found on neurons (N-type):

dominant form at presynaptic terminals• resistant to DHP antagonism• intermediate conductances, between T- and L-type• Subtypes can be distinguished by peptide toxins from

Pacific cone snails (Conus) and spiders (e.g., Agelenopsis aperta). Predatory invertebrate venoms have been purified and are commercially available, making channel ID easier.

• Subtypes: N (w-conotoxin GVIA sensitive), P/Q (w-Aga IVA; Purkinje cells), R (resistant), etc. (See Table 4.1 for a summary)

Genetics• transverse tubules in SkM are the first and richest known

source of L-type Ca channels• DHP served as a purification label• short AA seqs from a1 subunit were used to obtain a full-

length cDNA clone– 4 repeats of 6TM segments (including S4 and P regions) in each

repeat

• aux proteins: a2dbg

• cDNA library screens provided at least 10 mammalian variants

• Also, alternative splicing (small exons portions included or excluded), proteolytic cleavage of C-terminal tail, etc.

N

Permeation & Ionic Block• divalent cations strontium, barium, calcium (alkali

earth metals) all permeate Ca channels• Ba2+ yields the largest currents, and blocks K

channels too.• Vmcontrols r and

driving force

• [Ca]out = 104 to105 x [Ca]in, thus, outward ICa is miniscule, even at Vm more positive than ECa (e.g., +200mV). This is due to lack of ions.

‘Open-Channel’I-V for Ca

• model based on diffusion theory

• matches observation

• even though PK/PCa = 1/1000, IK rivals ICa

• this is due to relative scarcity of Ca2+, availability of K+ ions

[K]o = 2 mM[K]i = 100 mM[Ca]o = 2 mM[Ca]i = 100 nMECa = +124 mVreversal potential = observationequilibrium potential = calculated thermodynamicsErev ≠ Eequ! Why not?

More Outrageous Claims!• Even though Ca ions move down their EC gradient,

their movement differs from free diffusion:– ICa does not increase linearly with [Ca]o

– ICa is a saturating function of [Ca]o

– hence, maximum velocity for passing ions, “binding site” theory, and single file conduction: upper limit

• Ca channels become highly permeable to monovalent cations in the absence of divalent ones

• When Ca is present, at least one ion is “bound” within the channel at all times, preventing monovalent ions from entering

Inactivation

• LVAs (e.g., T-type) inactivate rapidly, and with Vm-dependence, while HVAs do not (at least not so quickly)• recall that [Ca]in below 100 nM is needed for maximal

responses, as if high [Ca]in blocks Ca channels• Brehm & Eckert (1978) and Tillotson (1979) remembered this

and suggested that free intracellular Ca blocks the channels somehow!• Thus, HVA function would be self-limiting, inactivating channels

until local [Ca]in is reduced via pumping

• In this scheme, inactivation is [Ca]in-dependent, and only indirectly Vm-dependent.

HVA (L-type) Inactivation• EGTA is a Ca chelator, and

is used as a buffer to keep [Ca]in low (fixed)• Ba2+ serves as a good

current carrier, but poor “inactivator”• little inactivation at very

large depolarizations, near ECa, where there is little ICa

2-Pulse Experiments• extent of inactivation ~

extent of Ca influx during pre-pulse• inactivation is maximal where

ICa is peak, not at peak depolarization

How does this compare to Na inactivation?

Note: this is not true for all Ca channels, but is true for HVA

pre-pulse:ICa = IBa

test pulse:ICa ≠ IBa

Molecular Mechanisms

HVA [Ca]in-dependent inactivation may be mediated by calmodulin molecules tethered to cytoplasmic regions (particularly, C-term) of the channel, bound with high affinity– inactivation disappears with mutation (deletion or

alteration) of calmodulin binding region– inactivation disappears if calmodulin is altered so

as not to bind Ca2+ (x4)

Activation: Delayed and Vm-dep

• rise of peak opening probability can be modeled by a Boltzmann relation with 3-5 gating charges moving across the membrane to control activation

• delay can be modeled by 2nd, 3rd, 5th, or 6th-order function of a probability particle (cf., m3 for Na, n4 for K)

• T-type act/inact has tm and th 20-50X longer than those for Na at same temp

• Closing (deactivation) begins immediately upon repolarization, with single or double exponential time course (tails), sometimes reflecting multiple types of ICa (T-LVA and L-HVA)

Single Channel Kinetics

• stochastic, as usualC ↔ C ↔ O

• opening viewed as 2-step process (e.g., m2), both steps Vm-dep• L-type channels can show sudden changes in gating

kinetics• typical sweeps: many sub-ms openings in 200 ms sweeps• other sweeps: longer channel openings, with k-2 100X slower

than typical sweeps• hence, “mode 0” (no opening) vs. “mode 1” vs. “mode 2”,

switching between the modes controlled at molecular level, and agonists stabilize mode 2, antagonists mode 0/1. ???

k1

k-1

k2

k-2

Review• permeating ions compete for binding sites within

the pore• Ca channels types differ in Vm-dep, inact, etc.• single channel gating described by state models

and chemical kinetics, which may change• drug-receptor sites change gating/mode, in Vm-dep

manner• Ca chs in all excitable cells, 2 roles:

– maintain inward current during prolonged depolarizations (no brisk inactivation)

– serve as ONLY LINK between depolarization and non-electrical activity

• axons: only important at terminals, ????• dendrites: electrical boosting, summation,

spreading of synaptic currents (t, l)• secretory/endocrine glands: slow inact allows for

prolonged depol and release• CardM & SmM: important electrical and

transducing roles during prolonged depol/contraction• Ca messenger (activity surrogate) affecting

replenishment, growth, and gene transcription•more to come!