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Snutch T P (2009) Voltage-Gated Calcium Channels. In: Squire LR (ed.) Encyclopedia of Neuroscience, volume 10, pp. 427-441. Oxford: Academic Press.

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Voltage-Gated Calcium Channels 427

Voltage-Gated Calcium Channels

T P Snutch , University of British Columbia, Vancouver, derived from a single gene product that is proteolyti- BC, Canada cally cleaved to form separate a2 and d subunits that

ancillary subunits surrounding the a1 subunit central Native Voltage-Gated Calcium Channels core. The entire complex appears to be roughly cylin- Calciumions are divalent cations that affect the elec- drical, with dimensions of approximately 9 nm wide by 20 nm high. The high-voltage-activated neuronal trical properties of cells and also act as second messengers regulating a variety of intracellular pathways. P/Q-type and N-type channels are also complexes con- This unique dual role is initiated by the rapid influx of sisting of an a1 subunit in association with one of four þ Ca2 through voltage-activated calcium channels in different b subunits (b1–b4)andoneoffoura2d subunits (a d –a d ). It is unclear whether there is an associated the plasma membrane which triggers the release of 2 1 2 4 neurotransmitters at nerve terminals, the initiation g subunit as part of native P/Q-type or N-type com- of muscle contraction, the regulation of intracellu- plexes. A general model has been proposed in which four or five proteins form the multisubunit high- lar signaling cascades and calcium-dependent gene expression, hormone secretion, and the control of voltage-activated calcium channel complex, although electrical excitability and firing patterns (Figure 1). subunit composition may differ depending on channel

The alteration of normal calcium channel function type (Figure 3). In the complex, the a1 subunit forms the channel proper, comprising the voltage-sensing and and intracellular calcium regulation are implicated 2þ in a number of human pathophysiological conditions, -gating mechanisms, the Ca -selective pore, and inter- including epilepsy, chronic pain, muscle dysfunction, action sites for drug binding and second messenger- dependent regulation. The ancillary proteins interact mood disorders, autism, and certain types of cardiovascular disease, cancer, and blindness. with the a1 subunit to modulate channel activity and Electrophysiological recordings from neurons, trafficking to the plasma membrane. The functional characteristics of the low-voltage-activated T-type cal- muscle, endocrine, and other cell types reveal multiple distinct types of calcium channels which differ in cium channels can be reconstituted by an a1 subunit their biophysical, pharmacological, and modulatory alone. properties. Generally, two major classes of calcium a1 (Cav) Subunits currents can be distinguished based on the membrane potentials at which they first open as well as other The large a1 subunit proteins (200–250 kDa) bear a biophysical properties (Figure 2). The low-voltage- high degree of similarity to the voltage-gated sodium activated calcium channels (also known as T-type) channel a1 subunits and are also evolutionarily transiently open in response to small changes from related to voltage-gated potassium channels and resting potentials, whereas high-voltage-activated cal- other members of the ion channel superfamily. The cium channels usually require stronger depolarizations mammalian genome contains 10 pore-forming cal- for activation. The high-voltage-activated channels are cium channel a1 (Cav) subunit genes grouped into a diverse class consisting of L-, N-, P/Q-, and R-types three interrelated subtypes (Figure 4). It is generally that canbe distinguished by their unique pharmaco- accepted that the distinct Cav subunits account for the logical, kinetic, and voltage-dependent gating charac- known types of native voltage-activated calcium teristics. Most electrically excitable cells express channels. Two branches of the Cav subunit phyloge- multiple subtypes of both low- and high-voltage- netic tree account for the high-voltage-activated activated calcium channels, the specific combination channels. In one branch, the Cav1.1–Cav1.4 subunits of which helps to define unique cellular phenotypes. all encode distinct L-type calcium channels, whereas The high-voltage-gated calcium channels are inte- a second branch Ca 2.1 encodes the P/Q-type, v gral membrane multisubunit protein complexes, with Cav2.2 the N-type, and Cav2.3 the R-type channel. the best biochemically characterized of these being The third Cav branch (Cav3.1–Cav3.3) encodes the skeletal muscle T-tubule-associated L-type chan- variants of low-voltage-activated T-type calcium nel. This L-type calcium channel consists of five channels. All of the Cav subunit genes are subject to distinct polypeptides designated a1, a2, b, d,andg extensive alternative splicing, resulting in multiple

(Figure 2). The a1, b,andg subunits are each derived functionally distinct isoforms of each of the 10 cal- from distinct genes, whereas the a2 and d subunits are cium channel subtypes.

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428 Voltage-Gated Calcium Channels

Ca2+

Plasma membrane

2+ Ca

Ca2+ Nerve and cardiac excitability, pacemaker activity Neurotransmitter release

Ca2+

Ca2+ 2+ 2+ Ca Ca

CaM Muscle contraction

Enzyme and allosteric CREB protein modulation Cell differentiation Gene regulation

Figure 1 Voltage-gated calcium channels mediate numerous physiological functions. In response to depolarizing changes in membrane potential, voltage-gated calcium channels rapidly open (within 1 ms), raising intracellular calcium to affect membrane electrical properties such as bursting and firing patterns as well as to regulate calcium-sensitive processes including muscle contraction, neurotransmitter release, cellular differentiation, and gene transcription.

þ The Ca v subunit structure is similar to voltage- over Na and electrostatically to a high rate of ionic activated sodium channels, consisting of four homol- flux. T-type calcium channels exhibit distinct perme- ogous domains (I–IV) that form the main integral ation characteristics, including a smaller pore diameter; membrane components of the channel (Figure 5). the ability to open with a half-size conductance level; 2þ 2þ The 10 Cav subunits share the most similarity in the and roughly similar permeation for Ca ,Ba ,and 2þ membrane-spanning portions of domains I–IV and Sr . The permeation differences between high-volt- the least similarity in the cytoplasmic linkers separat- age-activated and T-type channels are in part due to ing the domains and the cytoplasmic and amino distinct amino acids flanking the p-loops as well as to carboxyl tails. The highly divergent regions in the the domain III and IV p-loops of T-type channels pos- Cav subunits are the target sites of subtype-specific sessing aspartates rather than glutamate residues in second messenger-dependent modulation and other their selectivity filters. protein–protein interactions. Both high- and low-voltage-activated calcium

The permeation profile of the high-voltage-acti- channels intrinsically exhibit fast voltage-dependent þ þ þ vated calcium channels for cations is Ca2 > Ba2 > inactivation, a process that limits the amount of Ca2 þ þ þ þ þ þ Li > Na > K > Cs2 . Although Ca2 and Na entering cells during depolarization. Site-directed ions are of roughly similar diameter, the permeation mutagenesis and chimeric channel approaches have pathway of voltage-gated calcium channels is approxi- identified various specific amino acids and transmem- 2þ mately 1000-fold more selective for Ca than for brane segments of the Cav subunit as well as interac- þ b Na ions. The carboxylate side chains of four con- tions with subunits as being critical components of served p-loop glutamate residues within the lumen of inactivation. Overall, the rapid voltage-dependent the pore form the selectivity filter with a single high- inactivation of high-voltage-activated calcium chan- þ affinity Ca2 binding site. The four glutamate side nels is likely similar to that of both sodium and chains are not functionally equivalent and may flex to potassium channels and involves regions of the intra- þ enable a single Ca2 ion to bind with high-affinity or cellular mouth of the pore (S6 segments) being þ two Ca2 ions with lower affinity. This process would occluded by a movable intracellular segment in ‘ball- þ simultaneously contribute to both selectivity of Ca2 and-chain’- or ‘hinged lid’-type mechanisms. T-type

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Voltage-Gated Calcium Channels 429

1.0

0.8

0.6 T-type T-type HVA 0.4

0.2 Normalized conductance

200 pA

0.0 200 ms HVA −100 −80 −60 −40 −20 0 20 Voltage (mV)

Less depolarization required to open T-type Fast T-type channel inactivation channels (low threshold)

HVA

Slow T-type channel closing (deactivation) T-type

20 ms

Figure 2 High-voltage-activated (HVA) and low-voltage-activated (T-type) calcium channels exhibit distinct biophysical properties. T-type calcium channels can generally be distinguished by their more negative voltage dependencies of activation and inactivation, faster kinetics of inactivation, and slower kinetics of deactivation (closing).

calcium channel inactivation and activation are in dorsal root ganglion nociceptors and is required for strongly coupled, and the inactivation properties of both the mediation of basal thermal nociception and T-type channels can be distinguished as both very fast the development of thermal and mechanical hyperal- and calcium independent. Although many structure– gesia associated with inflammatory and neuropathic function-type studies have been reported, until a pain. Similarly, there appear to be pathophysiological calcium channel crystal structure becomes known, consequences associated with cardiovascular disease naturally occurring alternatively spliced variants pro- and alternative splicing that affect the biophysical vide both insightful structure–function information properties of Cav1.2 L-type channels. and clues toward how Mother Nature has altered spe- b Subunits cific channel regions to affect downstream physiological processes. For example, lengthening the Cav3.1 The b subunits are cytoplasmic proteins that directly T-type channel domain II–III and III–IV linkers through associate with the high-voltage-activated Cav subunits alternative splicing variously affects activation and to modulate channel trafficking and biophysical proper- b inactivation kinetics, recovery from inactivation, clos- ties. Structurally, the subunit proteins contain an ing rates, and the voltage dependence of both activation internal Src homology 3 (SH3) domain linked to an and inactivation. As another example, an alternative enzymatically inactive guanylate kinase (GK) domain splicing event that alters 14 amino acids in the proximal and are members of the membrane-associated GK carboxyl region of the Cav2.2 subunit affects N-type (MAGUK) family of scaffold proteins albeit with some current densities, electrophysiological properties, and distinct features. Analogous to other MAGUKs such as sensitivity to G-protein modulation. Furthermore, PSD-95, the calcium channel b subunit SH3 and GK one Cav2.2 splice variant is preferentially expressed domains exhibit a stable intramolecular interaction.

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430 Voltage-Gated Calcium Channels

Ca2+

S-S a 2

~140 kDa ~25 kDa g d a 1 ~ 30 kDa

~50–70 kDa ~200–250 kDa

Figure 3 Subunit composition of high-voltage-activated L-type calcium channel complex. Schematic diagram of the high-voltage- activated L-type calcium channel complex originally purified from skeletal muscle T-tubule membranes. The complex consists of five subunits: a1, a2, d, b, and g. The a1 subunit forms the channel proper, comprising the voltage-sensing and-gating mechanisms, the calcium-selective pore, and the target of most pharmacological agents. Functional T-type calcium channels consist of an a1 subunit alone, and there is no biochemical evidence indicating that other proteins are associated with native T-type channels in a multisubunit complex.

Cav1.3 protein from the endoplasmic reticulum to the plasma Cav2.1 Ca 1.4 membrane. On the a subunit, the Ca –b subunit v 1 v P/Q binding interaction occurs in a short cytoplasmic Cav2.2 Cav1.2 consensus motif in the domain I–II linker found in all

high-voltage-activated Cav subunits (termed the a1- N interaction domain (AID)). L There are four different b subunit genes in mam- Cav1.1 R Cav2.3 mals (b1–b4), each of which is subject to alternative splicing and each of which differentially affects the

T kinetic and voltage-dependent properties of high- voltage-activated Cav channels. The various b subunit isoforms vary significantly in primary sequence

in their N- and C-termini and in the linker separat- ing the SH3 and GK domains. The only available high-resolution structural information concerning Ca 3.1 Cav3.3 v voltage-gated calcium channel complexes is the Cav3.2 X-ray crystallographic definition of the interaction Figure 4 Similarity tree of the metazoan calcium channel a1 between the Cav domain I–II AID region and portions (Cav) subunit family. The amino acid sequences of representa- of the b subunit. The consensus motif of the Cav AID tives of the 10 metazoan calcium channel a1 subunits were aligned using Clustalx and the overall similarities are represented binds as an amphipathic helix to a deep groove of in the form of a similarity tree using HyperTree. the b subunit GK domain termed the AID-binding pocket.

Ca –b subunit binding occurs in 1:1 stoichiometry with v a d Subunits low nanomolar affinity to affect current amplitude, 2 kinetics, and the voltage dependences of activation The disulfide-linked a2 and d subunit proteins are and inactivation. The basis for affecting the level of heavily glycosylated, with the a2d subunit pre- calcium channel whole cell currents seems to be dominantly extracellular and anchored in the mem- through promoting translocation of the Cav subunit brane by a transmembrane segment formed by the

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Voltage-Gated Calcium Channels 431

a2d1 through a2d4 a1 (Cav) subunit Voltage sensor Pore loop g1 through g8

I II III IV

S-S

123456 1 2345 6 162345 162345

H N COOH 2 H2N COOH COOH H2N

COOH b through b 1 4

Figure 5 Schematic of the predicted structure and transmembrane topology of calcium channel complex proteins. Cav a1 subunits contain four homologous domains, each possessing six a-helical membrane-spanning segments (S1–S6) and a pore-forming p-loop. The S4 segment in each domain contains positively charged lysine or arginine residues at every third or fourth position forming a crucial part of the voltage-sensing mechanism of the channel. All high-voltage-activated channel Cav subunits possess a conserved EF hand motif in the C-terminus and also a conserved region in the domain I–II linker that binds in 1:1 stoichiometry to the cytoplasmic b subunit. The a and d 2 subunits are derived from a single gene product which is proteolytically cleaved to form the approximately 140 kDa a and approximately 2 d g 30 kDa subunits, which are then disulfide bonded to each other in the native state. The subunits are composed of four membrane- spanning regions with their C- and N-termini located intracellularly and with predicted extracellular N-glycosylation.

d subunit. Co-expression of a2d subunits with Cav g Subunits subunits affects current density, channel kinetics, and current–voltage relations, although generally There are at least eight g subunit genes in the mam- the a2d functional effects are more subtle that those malian genome (g1–g8), all of which are considered to for the b subunits. In mammals, there are four known be members of the Claudin family of tight junction- a2d genes, a2d1–a2d4. All four a2d subunit genes are associated adhesion molecules. Structurally, the subject to alternative splicing, with tissue and cellular C-termini of g2–g4, and g8 have a PDZ-binding specificity demonstrated for many of the variants. motif, whereas the analogous regions of g and g lack 7 5 In humans, the small organic molecules gabapentin this consensus motif but, rather,possess an SS/TSPC site and pregabalin are clinically effective anticonvul- likely designated toward specific protein interactions. sants that are also effective in neuropathic pain The g1 subunit is part of the skeletal muscle L-type conditions such as diabetic neuropathy, postherpetic calcium channel complex and is also found in heart, neuralgia, trigeminal neuralgia, and pain associated lung, spleen, kidney, liver, and testis. Mice lacking the with cancer and multiple sclerosis. Although syn- g1 subunit display altered skeletal muscle L-type cal- thetic analogs of the neurotransmitter g-aminobutyric cium currents, including a hyperpolarized shift in acid (GABA), gabapentin and pregabalin do not exert steady-state inactivation properties. Three other g their effects via interacting with GABA receptors or subunits, g2–g4, are of closest similarity to g1 and transporters but, rather, bind with high-affinity to the make up a subfamily of neuronal g subunits. high-voltage-activated calcium channel a2d1 and a2d2 Co-expression of some g subunits in combination subunits. Peripheral nerve injury in animals upregu- with Cav, b, and a2d subunits shows diverse and g lates a2d expression in the dorsal root ganglia and subtype-specific effects on peak current levels, kinet- spinal dorsal horn, suggesting that the a2d subunit ics, current–voltage relations, and steady-state inacti- contributes to central pain sensitization. Interest- vation properties. However, in other instances, g ingly, whereas a2d1 and a2d2 subunits associate with subunit co-expression does not appear to affect Cav high-voltage-activated calcium channel a subunits, properties, leaving open the question as to which g 1 including the L-type channels found in skeletal, subunit isoforms are bona fide components of high- smooth, and cardiac muscles, gabapentin and prega- voltage-activated calcium channel complexes. In one balin exhibit relatively few motor or cardiovascular intriguing instance, co-expression of g7 with Cav2.2 adverse effects at therapeutic doses. results in the complete abolition of N-type currents,

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432 Voltage-Gated Calcium Channels an effect apparently mediated by blocking expression T-type calcium channels are widely distributed rather than interfering with trafficking or channel to many central and peripheral neurons. At the biophysical properties. Additionally, a subset of g subcellular level, the Cav3.1, Cav3.2, and Cav3.3 iso- subunits (g –g , and g ) represent TARP family mem- forms differentially distribute to somatic and den- 2 4 8 bers (transmembrane AMPA receptor regulator pro- dritic compartments, implying that they uniquely teins) that regulate AMPA receptor trafficking as well contribute to neurophysiological processes. Outside as contribute to synaptic plasticity. the nervous system, T-type channels are found in

many excitable and nonexcitable cells, and evidence Low-Voltage-Activated (T-Type) Calcium indicates roles for T-type channels in pathophysiolog-

Channels: Ca 3.1–Ca 3.3 ical states such as tumor proliferation, renal injury, v v and cardiovascular disease (Tables 1–3).

The low-voltage activated (T-type) calcium channels T-type channels as a class generally lack selective, first activate at negative potentials (approximately high-affinity ligands. Small organic compounds 75 to 60 mV) and are distinctive in their fast and such as octanol, amiloride, and mibefradil have strongly voltage-dependent kinetics of activation been utilized as T-type channel antagonists, although and inactivation, rapid recovery from inactivation, they also variously inhibit high-voltage-activated slow closing, and small single channel conductances calcium channels and other conductances. Several

(Figure 2). Their hyperpolarized and overlapping classes of the clinically utilized antipsychotics and activation and inactivation ranges allow some cal- antiepileptics block T-type calcium channels, cium entry at resting membrane potentials (window although in most instances these agents are not current). This combination of unique biophysical specific (Figure 6). characteristics allows T-type calcium channels to gen- Ca 3.1 Subunit: CACNA1G Gene erally act as pacemakers, helping to trigger bursts of v sodium-dependent action potentials after membrane Cav3.1 channel biophysical properties are very simi- hyperpolarization. They also contribute to oscillatory lar to those of the Cav3.2 T-type, although Cav3.1 and rebound burst-firing behaviors relevant to normal channels do appear to exhibit a faster recovery from physiological functions, such as thalamocortical- inactivation when membrane potential returns to mediated deep sleep. Since T-type channels often negative values following depolarization. Cav3.1 sub- generate a resting inward current, they also likely con- units are expressed abundantly throughout the brain tribute to the gating of calcium-dependent ion chan- and the sinoatrial node of the heart, and lower levels nels and regulation of calcium-dependent enzymes are expressed in placenta, lung, and kidney. and gene expression. Mice in which the Cav3.1 gene is genetically deleted Although commonly referred to as a single class of appear anatomically and physiologically normal, channels, they are in fact physiologically and pharma- although there are several alterations to nervous cologically distinct T-type calcium channels encoded and cardiovascular system functions. Nervous system- by three different Cav subunit genes (Figure 4). All associated defects include an absence of GABAB recep- three Cav subunits (Cav3.1–Cav3.3) support strongly tor agonist-induced spike-wave discharges, a lack of voltage-gated calcium currents with the properties thalamic 1–4 Hz delta waves, a reduction of sleep expected of T-type channels, although they each spindles associated with normal sleep, and an enhanced exhibit distinct kinetic, permeation, pharmacological, visceral pain response. Overall, it appears that Cav3.1 and other voltage-dependent properties, most likely channels play a crucial role in thalamocortical burst- reflective of their unique primary sequences. Multiple firing processes relevant to a number of higher central Cav3 alternative splice variants have been identified nervous system functions, including thalamic sensory and probably also account for some of the observed gating and the control of consciousness. In the heart, heterogeneity in native T-type currents. The Cav3N- Cav3.1 gene deletion is associated with both slowing and C-termini and the intracellular linkers joining the of the heart rate (bradycardia) and atrioventricular transmembrane domains share little sequence identity dysfunction, indicating that this T-type calcium chan- with high-voltage-activated Cav subunits. Further- nel normally contributes to setting the basal heart more, Ca v3 subunit channels do not possess several rate and to atrioventricular conduction. specific functional motifs found in high-voltage-activated calcium channels, including the AID b subunit Cav3.2 Subunit: CACNA1H Gene binding site in the domain I–II linker and EF-hand/ calmodulin motifs in the C-terminus, the latter being Cav3.2 T-type channels are widely expressed in the þ consistent with the lack of Ca2 -dependent inactiva- central and peripheral neurons, cardiac tissue, kidney, tion for T-type channels. and liver. In dorsal root ganglion neurons, Cav3.2

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Voltage-Gated Calcium Channels 433

Table 1 Voltage-gated calcium channels

Native a1 Pharmacological sensitivity Major distribution Major cellular functions channel Subunit type

P/Q Cav2.1 o-Aga-IVA (P-type), Neurons, heart, pituitary Neurotransmitter release, o-CgTx-MVIIC (Q-type) Subcellular: cell bodies, dendrites, dendritic signaling

presynaptic terminals

NCa2.2 o-CgTx-GVIA, Neurons, neuroendocrine cells Neurotransmitter release, v o-CgTx-MVIIA Subcellular: subset of cell bodies, dendritic signaling, neurite dendrites, presynaptic terminals outgrowth LCa v1.1 Dihydropyridines, Cav1.1: skeletal muscle T-tubules Cav1.1: excitation–contraction benzothiazapines, coupling Cav1.2 phenylalkylamines Cav1.2: neurons, smooth (blood vessel, Cav1.2: excitation–contraction uterine, lung, stomach, intestine) and coupling, Ca2þ-dependent cardiac muscle; endocrine cells gene transcription

Cav1.3 Cav1.3: neurons, endocrine cells, heart Cav1.3: hormone secretion, þ atria, cochlea Ca2 -dependent gene transcription, atrial and

neuronal pacemaking activity

Ca 1.4 Ca 1.4: retina (photoreceptors, Ca 1.4: neurotransmitter release v v v horizontal, bipolar, and amacrine cells), in visual system immune system, low levels in spinal cord and skeletal muscle Subcellular: Cav1.2 and Cav1.3 on cell bodies and proximal dendrites of neurons; Cav1.4 at photoreceptor synapses

RCa v2.3 SNX-482 Neurons, heart Selective neurotransmitter Subcellular: cell bodies, some distal release, repetitive firing dendrites and presynaptic terminals

TCa3.1 Poorly defined–nonspecific Neurons, smooth muscle, endocrine Pacemaking activity, repetitive v block by mibefradil, cells, placenta, testis, lung, kidney spiking and bursting, dendritic

amiloride, pimozide, cardiac sinoatrial cells, neuroendocrine neurotransmitter release, 2þ 2þ 2þ flunarizine, Ni ,Zn ,Cd cells, spermatogenic cells, developing cellular differentiation skeletal muscle, fibroblasts, osteoblasts, astrocytes Cav3.2 Subcellular: soma, proximal and distal dendrites

Cav3.3

channels contribute to both acute and neuropathic absence epilepsy (CAE) and idiopathic generalized pain behaviors, whereas in mechanoreceptors they epilepsy (IGE). The majority of the mutations are are required for the transduction of slow-moving localized in the Cav3.2 channel intracellular linker mechanical stimuli. In sperm, the activation of Cav3.2 between domains I and II, and biophysical analyses channels is required for triggering the acrosome reac- reveal both gain-of-function and loss-of-function tion and allowing sperm to become competent for effects. The gain-of-function effects are predicted to fusion with oocytes. Pharmacologically, Cav3.2 chan- result in increased calcium influx at lower membrane nels can be distinguished from both Cav3.1 and Cav3.3 potentials, which would result in neuronal hyperex- by the higher sensitivity to nickel blockade (IC50 of citability and increased spike-wave discharge. The 10 mMvs. 200 mM). polygenic, non-Mendelian inheritance of CAE and Genetic deletion of Cav3.2 in mice results in smal- IGE suggests that interaction with other gene pro- ler but viable animals that possess constricted ducts must also be considered with regard to disease coronary arteries and develop cardiac fibrosis. In pathogenesis. agreement with the high levels of Cav3.2 channels in Missense mutations in the CACNA1H gene result- small dorsal root ganglion cells, knockout mice also ing in a predicted overall decrease in calcium entry exhibit attenuated responses to mechanical, thermal, through Cav3.2 channels have also been associated and chemical pain stimuli. with a small subset of individuals with autism spec- Point mutations in the CACNA1H gene in humans trum disorder (ASD). Considering the tight regulation are associated in a subset of patients with childhood of intracellular calcium levels necessary for normal

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434 Voltage-Gated Calcium Channels

Table 2 Spontaneous mutations in mammalian calcium channel subunit genes

Gene and human Channel Disease state/phenotype chromosomal location subunit

CACNA1S 1q31–32 Cav1.1 Human (dominant mutations): hypokalemic periodic paralysis-periodic muscle weakness; malignant hypothermia susceptibility type 1-elevated skeletal muscle contraction, glycogenolysis, increased heat and lactic acid acidosis, tachycardia, and cardiac arrhythmia and arrest

Mouse: recessive mutations, muscular dysgenesis (mdg)

CACNA1C 12p13.3 Cav1.2 Human: Timothy syndrome–severe functional and developmental abnormalities in several organ systems, including heart, skin, eyes, teeth, immune system, and the brain; autism

CACNA1F Xp11.23 Cav1.4 Human (recessive and dominant mutations): incomplete X-linked congenital stationary night blindness–variable degrees of night blindness, low visual acuity, nystagmus, and hyperopia or myopia; X-linked cone–rod dystrophy–low visual acuity, reduced color vision, central scotomas in visual field, photophobia, and myopia

CACNA1A 19p13.1 Cav2.1 Human (dominant mutations): episodic ataxia type 2–spontaneous episodes of ataxia, nystagmus, and often migraine-like symptoms; familial hemiplegic migraine type 1– migraine with aura, motor aura typically manifests as hemiplegia in upper and lower

extremities, often associated with ataxia or nystagmus; spinal cerebellar ataxia type 6–

gate ataxia, reduced coordination, nystagmus, dysarthia and proprioceptive sensory loss, severe cerebellar Purkinje cell loss la Mouse (recessive mutations): tottering (tg), leaner (tg )–ataxia, dystonia, spike-wave epilepsy; dominant mutation; wobbly ataxia CACNA1H 16p13.3 Cav3.2 Human: idiopathic generalized epilepsy/childhood absence epilepsy–absence seizures including short seizures followed by sudden impairment of consciousness; autism spectrum disorder Rat (recessive mutation): absence epilepsy

CACNA2D a2–d2 Mouse: recessive mutation; ducky–spike-wave epilepsy

CACNB4 2q22-23 b4 Human: generalized epilepsy–recurrent generalized seizures, episodic ataxia Mouse: lethargic (lh)–spike-wave epilepsy

CACNG2 g Mouse (recessive mutations): stargazer (stg); waggler (stgwag), altered skeletal muscle 2 L-type current, spike-wave epilepsy

Table 3 Targeted gene deletions in mouse calcium channel subunit genes

Gene Channel Disease state/phenotype subunit

CACNA1S Cav1.1 Nonviable, suffocate at birth due to inability to contract diaphragm CACNA1C Cav1.2 Die approximately embryonic day 12.5 CACNA1D Cav1.3 Viable, deaf, atrial arrhythmias, bradycardia CACNA1F Cav1.4 Viable, impaired vision CACNA1A Cav2.1 Usually die approximately 3 weeks postnatal, absence seizures, ataxia, dystonia, spasms, abnormal cerebellar synapses and cerebellar cell death, altered acute and inflammatory pain responses

CACNA1B Cav2.2 Viable, reduced neuropathic and inflammatory pain responses, altered responses to ethanol and anesthetics, hyperactive, increased basal vigilance state, reduced baroreflex, decreased anxiety, impaired long-term memory

CACNA1E Ca 2.3 Viable, altered visceral pain response, impaired spatial memory and locomotion, altered responses v to cocaine, anesthetics and cerebral ischemic damage, decreased insulin response and glucose tolerance CACNA1G Cav3.1 Viable, absence of GABAB receptor agonist-induced spike-wave discharges, decreased heart rate, slower atrioventricular conduction CACNA1H Cav3.2 Viable, impaired relaxation of vasculature, altered acute and chronic pain responses CACNB1 b1 Perinatal lethality, no excitation–contraction coupling CACNB2 b2 Embryonic lethal CACNB3 b3 Viable, altered blood pressure in response to high-salt diet, reduced acute and inflammatory pain responses

CACNB4 b4 Ataxia, focal motor seizures, absence epilepsy

CACNG1 g1 Viable, no visible phenotype CACNG4 g4 Viable, no visible phenotype

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Ethosuximide (T-type) Ziconotide (N-type)

Gabapentin, pregabalin S-S a2

g a d 1

Flunarizine, pimozide (T-type)

Dihydropyridines, phenylaklamines, benzothiazepines (L-type) Figure 6 Clinical agents utilized to treat human cardiovascular and neurological disorders. The L-type calcium channel antagonists

(dihydropyridines, phenylalkylamines, and benzothiazepines) block all Cav1 subunits in vitro, but they predominantly act in vivo on the cardiovascular system and exhibit selectivity between cardiac and vascular functions. Gabapentin and pregabalin bind to specific arginine residues in the a2d1 and a2d2 subunits, but determination of the exact mechanism of action of gabapentin has proven elusive, with reports both supporting and refuting direct actions on high-voltage-activated calcium channel properties. Ziconotide is a 25-amino acid synthetic version of o-contotoxin MVIIA that reversibly blocks N-type channels and is highly effective in multiple types of human chronic and neuropathic pain conditions. Side effects of ziconotide administration include orthostatic hypotension, confusion, nystagmus, sedation, auditory and visual hallucinations, and agitation. Clinical antipsychotics such as pimozide and flunarizine block T-type channels, but they also target dopamine receptors. Ethosuximide, an absence epilepsy therapeutic, blocks T-type channels at millimolar concentrations.

cell signaling and gene expression, it is possible that types of non-excitable cells (Table 1). In cardiac, the biophysical effects of the CACNA1H mutations skeletal and smooth muscles they are the primary translate into neuronal development abnormalities. route for calcium entry and are responsible for trig- Similar to that for CAE and IGE, the strong polygenic gering muscle contraction. In neurons, L-type chan- inheritance pattern of ASD, together with the fact nels are primarily located on cell bodies and proximal that many patients do not have associated mutations dendritic regions where they are vital players in link- in CACNA1H, indicates a likely minor role for the ing electrical excitability to the control of calcium- Ca 3.2 channel in overall ASD susceptibility. dependent gene transcription. Some L-type channels v are also involved in exocytotic release from both Cav3.3 Subunit: CACNA1I Gene endocrine cells and specialized neurons such as photoreceptors. Compared to the Cav3.1 and Cav3.2 T-type channels, L-type calcium channels generally require large recombinant Cav3.3 channels uniquely exhibit voltage- dependent facilitation, a larger time constant of activa- depolarizations to become activated, typically opening at potentials positive to 30 mV, although they tion (slower opening), as well as a slower rate of inactivation and a faster deactivation (closing). Together, can activate at significantly more negative potentials in chromaffin cells, sensory neurons, photoreceptors, these properties predict that Cav3.3 channels generate sustained inward calcium current under conditions and cardiac cells. In addition to voltage-dependent mechanisms, L-type currents inactivate rapidly of prolonged depolarizations as well as a large window þ through a Ca2 -dependent negative feedback process calcium current under resting conditions. Also com- þ that serves to finely control Ca2 -dependent signaling pared to Cav3.1 and Cav3.2 channels, Cav3.3 T-type þ and protect against toxic Ca2 overload. channels are differentially modulated by second messenger signaling pathways, including those acting L-type calcium channels are readily distinguished by through muscarinic acetylcholine and metabotropic their sensitivities to several classes of small organic glutamate receptors. molecules widely used in the clinical treatment of hypertension, angina, Raynaud’s disease, and cerebral

vasospasm. The 1,4-dihydropyridines (DHPs), phenyl- High-Voltage-Activated Calcium alkylamines, and benzothiazepines interact at alloste- Channels rically distinct binding sites on the L-type Ca subunit v to block channel activity. The in vivo specificity of L-Type Calcium Channels: Cav1.1–Cav1.4 physiological actions of these agents likely reflects a High-voltage-activated L-type calcium channels are number of factors, including pharmacokinetic aspects, expressed in almost all excitable cells as well as many the existence of distinct L-type molecular isoforms

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436 Voltage-Gated Calcium Channels

þ (Ca 1.1–Ca 1.4), and the state dependence of drug requiring Ca2 influx through Ca 1.2 channels to v v v interaction. The state dependence of channel blockade activate RyR2 receptors to release calcium from inter- þ is of particular relevance since both drug access to nal SR Ca2 stores in order to initiate muscle contrac- its binding site and the affinity of drug binding are tion. In the cardiovascular system, Ca 1.2 channels are v highly dependent on the conformational state of the critically linked to vasodilation and cardiac depression channel. Several DHP agonists have also been devel- and are the target of the clinically used L-type channel oped, the most highly utilized being (-)-Bay K 8644, blockers. Cardiovascular Cav1.2 L-type channels are which increases both L-type channel open time and also a target for modulation by noradrenaline and single channel conductance. angiotensin II, both of which are critical regulators of vascular control. Within the brain, Cav1.2 is the major Cav1.1 Subunit: CACNA1S Gene L-type channel, with Ca 1.2 channels being diffusely v Cav1.1 L-type calcium channels are crucial in striated distributed at low levels on cell bodies and proximal muscle for coupling membrane depolarization to the dendrites. Multiple alternatively spliced Cav1.2 variants have been identified that exhibit distinct bio- release of calcium from cytoplasmic stores which then triggers excitation–contraction (EC) coupling. Depo- physical properties, including rates of activation and larizing changes in skeletal muscle membrane poten- current–voltage relations. Many Cav1.2 splice varia- tial cause a conformational change in Ca 1.1 which tions are localized to the cytoplasmic linker between v induces an allosteric interaction with the sarcoplas- domains II and III and the carboxyl region, likely mic reticulum (SR) ryanodine receptor (RyR1), ulti- reflecting cell-specific protein interactions and physio- matelytriggering calcium release from the SR and logical functions. Interestingly, some splice variants exhibit cell-, tissue-, and pathophysiological state- subsequent muscle contraction. Unique in this process is the fact that calcium entry through the Cav1.1 specific expression patterns. Reflective of its physio- channel is not required for muscle contraction; rather, logical importance in cardiac and nervous system development, genetic deletion of Ca 1.2 in mice is the Cav1.1 channel complex acts simply as a voltage v sensor to initiate the contraction process. lethal and animals die on approximately embryonic Related to its role as a voltage sensor for the RyR1, day 12.5. the Ca 1.1 L-type channel is implicated in two muscle Mutations in the domain I S6 transmembrane seg- v disorders in humans: hypokalemic periodic paralysis ment of the human Cav1.2 gene are associated with a (HypoPP) and malignant hyperthermia susceptibi- serious multisystem disorder called Timothy syn- lity (MHS; Table 2). Missense mutations in the drome (TS). More than half of TS patients die between 2 and 3 years of age. Functional analysis of CACNA1S gene encoding Cav1.1 result in reduced current density and reduced rates of activation, loss- TS Cav1.2 mutations shows a near complete loss of of-function effects predicted to result in less calcium voltage-dependent inactivation and altered CaMKII- influx into muscle cells during depolarization. dependent channel gating, both of which are pre- 2þ Although reduced calcium influx through Cav1.1 dicted to prolong Ca influx. Computer simulation channels could indirectly affect membrane potential, of these effects suggests significant cardiac action given the dual functions of Ca 1.1 it is also possible potential prolongation and prolonged QT intervals, v that the pathophysiology of HypoPP is independent a phenomenon known to result in abnormal second- þ of Ca2 flux but is associated with an uncoupling of ary depolarizations, arrhythmia, and sudden death.

EC between Cav1.1 and RyR1 and indirectly results þ in reduced Ca2 release from internal stores. Cav1.3 Subunit: CACNA1D Gene MHS is an autosomal dominant disorder characterized by a predisposition in otherwise healthy indi- Cav1.3 L-type channels have sometimes been termed ‘neuroendocrine’ L-type channels because of their viduals for muscle hypermetabolism in response to exposure to volatile anesthetics or depolarizing mus- presence in neuroendocrine cells, such as pancreatic cle relaxants. The majority of patients would die b and adrenal chromaffin cells. However, they are without early administration of the SR calcium also found in the brain, cardiac atrial myocytes, ret- release inhibitor dantrolene. Most mutations asso- ina, ovaries, and cochlear hair cells of the ear. Within ciated with MHS are in the RyR1 gene, although the central nervous system, Cav1.3 channels are several have also been identified in CACNA1S. found in most regions, although they are generally less abundant than Ca 1.2 L-type channels. In con- v trast to the Ca 1.1 and Ca 1.2 L-types, Ca 1.3 cur- Ca 1.2 Subunit: CACNA1C Gene v v v v rents possess more negative current–voltage relations In both cardiac and smooth muscles, Cav1.2 L-type and have a lower sensitivity to block by dihydro- channels are involved in an EC coupling mechanism pyridines. In adult substantia nigra neurons, the

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Voltage-Gated Calcium Channels 437 more hyperpolarized activation of Cav1.3 channels CACNA1F gene and is predicted to cause either a 2þ contributes to the autonomous Ca -dependent pace- premature stop codon or variable size deletions. The maker activity of these cells. Interestingly, the inhibi- functional consequences of this mutation are unknown. tion of substantia nigra pacemaking activity by the P/Q-Type Channel – Ca 2.1 Subunit: pharmacological blockade of Ca 1.3 channels is pro- v v CACNA1A Gene tective in mouse models of Parkinson’s disease. Cav1.3 gene-deficient mice are largely anatomically P/Q-type calcium channels are one of the most abun- and behaviorally normal, with notable exceptions. dantly expressed calcium channel subtypes in the (1) The inner hair cochlea cells completely lack L-type mammalian central and peripheral nervous systems. currents, which results in deafness, and (2) sinoatrial Cav2.1 channels are highly localized at presynaptic node dysfunction results in atrial arrhythmias and terminals throughout the brain, spinal cord, and neu- bradycardia, effects reflective of the negative activation romuscular system, where they mediate calcium of Cav1.3 channels toward contributing to cardiac influx essential for a large portion of excitatory neu- pacemaking activity and diastolic depolarization. rotransmitter release. At postsynaptic sites, Ca 2.1 v P/Q-type channels contribute to the precise modula- Ca 1.4 Subunit: CACNA1F Gene tion of intracellular calcium levels important for den- v dritic firing and also second messenger signaling Ca 1.4 currents are unique in their small unitary v relevant to calcium-dependent gene transcription. conductance, slow inactivation kinetics, and lack of Originally defined as separate channel types, P-type þ Ca2 -dependent inactivation. Similar to that for the and Q-type channels differ in their relative sensitiv-

Cav1.3 L-type, Cav1.4 channels activate at relatively ities to the spider peptide toxin o-agatoxin IVA and negative potentials and are predicted to support a the cone snail toxin o-conotoxin MVIIC, and they substantial resting window current. also display distinct time- and voltage-dependent In contrast to the rapid and transient nature of properties. In fact, the single Ca 2.1 subunit gene v neurotransmitter release at most neuronal synapses, encodes both P- and Q-type channels with their dis- synaptic release from photoreceptors occurs through tinctive properties reflecting small protein alterations graded changes in membrane potential and is depen- generated by alternative splicing. There may also be dent on Ca 1.4 L-type calcium channel activity. In the v some contribution to the phenotypic differences dark, photoreceptors tonically release glutamate, between P-type and Q-type currents by the inclusion whereas light stimulus causes membrane hyperpolari- of different b subunit isoforms to form distinct chan- zation and the termination of glutamate release. The nel complexes. lack of calcium- and time-dependent inactivation Deletion of the Cav2.1 subunit gene in mice results together with the occurrence of a large window cur- in animals with largely normal embryonic and early rent make Cav1.4 channels ideally suited to support postnatal development but with the onset of severe tonic glutamate release from photoreceptors. In sup- neurological complications starting on approximately port of this notion, mutations in the CACNA1F gene postnatal day 10 and resulting in the death of most, are implicated in the vision-related channelopathies, but not all, animals by approximately postnatal day incomplete X-linked congenital stationary night 21 (Table 3). In surviving animals, cerebellar defects blindness (IXLCSNB) and X-linked cone–rod dystro- such as Purkinje cell swelling and abnormal granule phy (CORDX). cell migration are present. More than 60 missense and truncation mutations Mutations in the human CACNA1A gene are asso- scattered across most Cav1.4 structural regions have ciated with severe neurological channelopathies, been identified in the CACNA1F gene of patients including familial hemiplegic migraine (FHM1), epi- with IXLCSNB. Biophysical investigation of some sodic ataxia type 2 (EA2), and spinocerebellar ataxia mutations indicates no obvious measurable affect on type 6 (SCA6). FHM1 is a rare autosomal dominant Cav1.4 channel properties, whereas others cause subtype of migraine with an aura phase linked to corti- either a loss of function or a gain of function. Overall, cal spreading depression (CSD). Mutations in CAC- the analysis of IXLCSNB mutations to date does not NA1A resulting in FHM1 are primarily localized to give a clear or consistent indication regarding how Cav2.1 channel S4 voltage sensors and flanking regions. the distinct point mutations might result in abnormal Phenotypic dissection of these mutations in isolation neurotransmission in photoreceptors. has proven difficult and may be due to the fact that

CORDX is a progressive disorder including clinical distinct Cav2.1 alternatively spliced variants are differ- features of reduced visual acuity and poor color entially affected by FHM1 mutations. More revealing vision. In one CORDX family, a mutation has been data from mice carrying introduced FHM1 mutations identified in the splice acceptor site of intron 28 in the predict a P/Q-type channel gain-of-function phenotype

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438 Voltage-Gated Calcium Channels resulting in a lower threshold for CSD depression and sensitivity to irreversible block by o-conotoxin an increased velocity of propagation across the cortex. GVIA, a 27-amino acid peptide from the marine It is unknown how headache pain is manifest, although hunting snail Conus geographus. one possibility is that CSD activates trigeminovascular N-type channels are distributed throughout the afferents and evokes meningeal and brain stem activity peripheral and central nervous systems and appear consistent with migraine pain. It is also possible that especially concentrated in regions of high synaptic

Cav2.1 P/Q-type channels directly contribute to modu- density where they are responsible for triggering lation of the trigeminal pain pathway. neurotransmitter release at a subset of terminals. In EA2 is an autosomal dominant disorder in which addition to their presynaptic functions, N-type chan- patients experience spontaneous episodes of ataxia nels are also localized postsynaptically on dendrites, lasting from hours to days. Approximately half of where they likely contribute to the integration and patients also experience migraine-like symptoms. amplification of neural inputs. They also appear to EA2 is genetically variable with more than 40 indi- play a role in nervous system development and neur- vidual missense, truncation, and alternative splice site ite outgrowth. mutations distributed throughout the channel. Some Although no natural mutations in the CACNA1B truncation and missense mutations cause a reduction gene have been described, Cav2.2 gene knockout and in current density, possibly due to fewer channels pharmacological interventions indicate that the being properly folded and reaching the plasma mem- N-type calcium channel plays a critical role in the brane. Alternatively, some missense mutations show a primary afferent nociceptive pathway. N-type cal- net reduction of available channels due to a depolar- cium channels are highly concentrated in the cell izing shift in the voltage dependence of activation, bodies and synaptic terminals of a subset of primary an increased rate of inactivation, and a reduced afferents that terminate in the dorsal horn of the rate of recovery from inactivation. Overall, all EA2 spinal cord (mainly C-fibers and A-d fibers). Deletion mutations exhibit a net reduction in Ca 2.1-mediated of the Ca 2.2 channel gene in mice results in animals v v calcium currents, likely resulting in reduced neuro- largely resistant to the induction of neuropathic and transmitter release at critical synapses. inflammatory pain but otherwise exhibiting normal Patients with SCA6 have a polyglutamine (CAG) sensory and motor functions (Table 3). Pharmacolo- expansion in exon 47 of the CACNA1A gene, making gically, the marine snail C. magus peptide toxin, SCA6 a member of the group of neurodegenerative o-conotoxin-MVIIA (also known as ziconotide), disorders containing CAG repeats and which includes reversibly blocks N-type currents and has been widely Huntington’s disease (Table 2). Unaffected people have used to define the contributions of N-type channels a low copy number of CAG repeats (4–16), whereas in both animal model and human pain conditions. patients with SCA6 have expansions of more than 21 The intrathecal administration of ziconotide in ani- CAG repeats. The length of the expansion appears to mals shows strong analgesic effects on inflammatory be directly correlated with age of onset, with the greater pain, postsurgical pain, thermal hyperalgesia, and CAG expansion being associated with early age of mechanical allodynia. In humans, the intrathecal disease onset. Biophysical analyses of polyglutamine administration of ziconotide to patients unresponsive expansions in the Cav2.1 calcium channel show a to intrathecal opiates significantly reduces pain scores range of effects on voltage- and time-dependent pro- in chronic conditions such as cancer and HIV-related perties with a strong dependence on both auxiliary pain. Ziconotide has also been shown to be effective subunitand Ca 2.1 subunit splice variant composition. for the management of intractable spasticity follow- v ing spinal cord injury. N-Type Channel – Ca 2.2 Subunit: CACNA1B Gene v Other behavioral phenotypes associated with dele-

Unique among the high-voltage-activated calcium tion of the Cav2.2 gene in mice include a decrease in channels, the expression of N-type channels is largely both the voluntary consumption and the hypnotic restricted to neurons and neuroendocrine-derived cells effects of ethanol and the accentuation of ethanol- (Table 1 ). Biophysically, N-type channels exhibit con- based place preference, decreased anxiety, reduced ductances intermediate between that of T-type and that baroreceptor response, and impaired long-term of L-type channels and display time-dependent inacti- memory (Table 3). vation slower than the rapid inactivation of T-type R-Type Channel – Ca 2.3 Subunit: CACNA1E Gene channels but faster than that of L-type channels. In v some cells, such as sympathetic neurons, N-type cur- A component of high-voltage-activated calcium currents do not always inactivate rapidly and the decay rent remains in some neurons even after the applica- rate can be slow and incomplete. Pharmacologically, tion of pharmacological agents to block L-, N-, and N-type channels can be distinguished by their high P/Q-type channels. Categorized as R-type for residual

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Voltage-Gated Calcium Channels 439 or resistant, this current can be blocked by the of the Ca 2.3 subunit gene appear anatomically v African tarantula venom peptide SNX-482. Although normal but exhibit a number of physiological defects, it is possible that a portion of the native R-type cal- including increased anxiety, decreased spatial memory cium current as defined pharmacologically may result and locomotor activity, altered sperm motility, and from incomplete blockade by the applied antagonists decreased insulin secretion and glucose tolerance. and/or to the existence of pharmacologically distinct Pain responses are also affected, with a decrease in splice variants of the P/Q- and N-type channels, the acute formalin-induced inflammatory pain but an

Cav2.3 subunit can account for many native R-type increased long-term response to formalin-induced properties. visceral pain. In some instances, the voltage-dependent para- meters of Ca 2.3 channels (current–voltage relation- v Regulation of Calcium Channel ship and voltage dependence of inactivation) are more Activity – Interacting Proteins hyperpolarized than those of the other high-voltage- activated channels, and Cav2.3 channels also appear The activities of all calcium channel subtypes are 2þ 2þ unique in being equally permeable to Ca and Ba highly regulated by intracellular second messengers, 2þ and being highly sensitive to Ni blockade, properties which has significant consequences concerning down- which are usually ascribed to T-type calcium channels. stream phy siologi cal funct ions (Figure s 7 and 8). For example, the modulation of presynaptic P/Q-type and The exact physiological functions of R-type channels are the least defined of the high-voltage-activated N-type channels by G-protein-coupled receptors subtypes. Although N- and P/Q-type channels domi- (GPCRs) is a common strategy for regulating synaptic nate neurotransmitter release, at some synapses R-type strength and efficacy. Many GPCRs, including those channelsalso contribute to release. Mice with deletion for dopamine, opioids, neuropeptide Y, glutamate,

Ca2+

Nt Nt

GPCR GPCR b a a b TK g g b

PKA CaMKII as

cAMP CaM a o/i PTX

PKG

PKC DG aq cGMP

ER IP3

Figure 7 Second messenger-dependent modulation of calcium channels. The functional activities of all high-voltage-activated and low- voltage-activated T-type calcium channels are subject to modulation (upregulation and downregulation) by the activation of intracellular second messenger pathways acting via most known G-protein-coupled receptors (GPCR). DG, diacyl glycerol; ER, endoplasmic reticulum; IP3, inositol triphosphate; NT, neurotransmitter; PTX, pertussis toxin; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; TK, tyrosine kinase. Redrawn from Chemin J, Traboulsie A, and Lory P (2006) Molecular pathways underlying the modulation of T-type calcium channels by neurotransmitters and hormones. Cell Calium 40: 121–134.

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440 Voltage-Gated Calcium Channels

I II III IV

162345 1 2345 6 162345 162345

EF hand H2N

IQ Pre-IQ CBD : Gbg

: Skeletal EC-coupling COOH : AKAP-79

: CSN5/Jab1 : Calmodulin : Cysteine string protein : CaM kinase II : Syntaxin-1 : Ca-binding protein-1 : SNAP-25 : CASK : Synaptotagmin-1 : Mint-1

: Sorcin

: VILIP-2 : AKAP-15

: Homer-1 (cardiac/smooth muscle EC-coupling)

Figure 8 Voltage-activated calcium channels interact with multiple types of intracellular signaling molecules. Structure–function and biochemical analyses indicate that multiple types of intracellular proteins bind to and modulate calcium channel activity, trafficking, and interactions with intracellular signaling proteins. These include a high-affinity G-protein bg subunit binding, distinct regions involving functional interactions with the synaptic release machinery (SNAP-25, syntaxin-1, and synaptotagmin), binding to anchoring proteins (AKAP-79 and AKAP-15), interactions with the skeletal and cardiac muscle excitation–contraction coupling machinery, as well as binding 2þ with regulatory molecules such as calmodulin, CaM kinase II, Ca -binding protein-1, CASK, and mint-1.

GABA, substance P, canabinoids, adenosine, and estradiol, GABA, serotonin, somatostatin, substance P, somatostatin, all act to attenuate calcium presynaptic and norepinephrine. T-type current upregulation channel activity and to thereby decrease neurotrans- includes pathways acting via acetylcholine, aldoste- mitter vesicle fusion and release. rone, angiotensin II, ATP, L-cysteine, erythropoietin,

In another prominent example of calcium channel estrogen, GABA, serotonin, norepinephrine, vasoac- þ modulation, calmodulin acts in a Ca2 -dependent feed- tive intestinal peptide, insulin-like growth factor, and þ back mechanism to either inhibit (Ca2 -dependent inhi- substance P. Altering T-type channel activity is pre- 2þ bition (CDI)) or facilitate (Ca -dependent facilitation dicted to critically affect neuronal and cardiac physio- (CDF)) high-voltage-activated calcium activity. CDF logical processes such as firing patterns, rhythmic and CDI are mechanistically linked, predominantly oscillations, and pacemaker activities. involving several regions of the Cav subunit carboxyl Presynaptic P/Q-type and N-type channels provide tail, including an EF-hand motif and several down- the initial calcium trigger to initiate neurotransmitter þ stream regions that form calmodulin (CaM), Ca2 - release and contribute to anchoring synaptic vesicles binding protein, and CaM kinase II tethering/binding at release sites. Direct interaction occurs between the sites. synaptic protein interaction site in the domain II–III T-type channel activity can also be upregulated or linker of P/Q-type and N-type channels and the downregulated by many types of neurotransmitter SNARE proteins syntaxin-1A and SNAP-25. The and hormone receptor-coupled pathways. T-type cur- Cav2.1 channel subunit also binds to the synaptic rent inhibitory pathways include those mediated via calcium sensor protein, synaptotagmin. Disruption acetylcholine, endocannabinoids, angiotensin II, atrial of the Cav subunit–SNARE protein interaction in natriuretic factor, bradykinin, dopamine, enkephalin, neurons inhibits neurotransmitter release.

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Voltage-Gated Calcium Channels 441

There are examples in which parts of individual Gomez-Ospina N, Tsuruta F, Barreto-Chang O, Hu L, and calcium channel complexes perform roles other than Dolmetsch R (2006) The C terminus of the L-type voltage-gated calcium channel Ca 1.2 encodes a transcription factor. Cell 127: v mediating calcium influx or voltage-sensing activities. 591–606.

In one instance, a b4 subunit splice variant indepen- Hering S, Berjukow S, Sokolov S, et al. (2000) Molecular determi- 2þ dently and directly interacts with a nuclear protein nants of inactivation in voltage-gated Ca channels. Journal implicated in gene silencing and transcription. In of Physiology 528: 237–249. Hudmon A, Schulman H, Kim J, Maltez JM, Tsien RW, and Pitt GS another instance, the b3 subunit appears to affect þ (2005) CaMKII tethers to L-type Ca2 channels, establishing a þ glucose-mediated excitability in pancreatic cells by local and dedicated integrator of Ca2 signals for facilitation. negatively affecting inositol 1,4,5-triphosphate- Journal of Cell Biology 171: 537–547. induced calcium release. Furthermore, an approxi- Khosravani H and Zamponi G (2006) Voltage-gated calcium chan- mately 75 kDa proteolytically cleaved C-terminal nels and idiopathic generalized epilepsies. Physiological Reviews 86: 941–966. fragment of Cav1.2 L-type channel can translocate Llinas R and Yarom Y (1981) Electrophysiology of mammalian to the nucleus, where it interacts with a nuclear pro- inferior olivary neurons in vitro. Different types of voltage- tein to regulate the transcription of multiple neuronal dependent ionic conductances. Journal of Physiology 315: genes. 549–567. Mangoni ME, Couette B, Marger L, Bourinet E, Striessnig J, and See also: Calcium Channels; Calcium Channel and Nargeot J (2006) Voltage-dependent calcium channels and Calcium-Activated Potassium Channel Coupling; cardiac pacemaker activity: From ionic currents to genes. Prog- ress in Biophysics and Molecular Biology 90: 38–63. Neuromodulation of Calcium Channels; Voltage Gated Peres-Reyes E (2002) Molecular physiology of low-voltage- Potassium Channels: Structure and Function of Kv1 to activated T-type calcium channels. Physiological Reviews 83: Kv9 Subfamilies; Voltage-Gated Potassium Channels 117–161. (Kv10–Kv12). Pietrobon D (2005) Function and dysfunction of synaptic calcium channels: Insights from mouse models. Current Opinion in

Neurobiology 15: 257–265.

Further Reading Proenza C, O’Brien J, Nakai J, Mukherjee S, Allen PD, and Beam KG (2002) Identification of a region of RyR1 that parti- Carbone E, Giancippoli A, Marcantoni A, Guido D, and Carabelli cipates in allosteric coupling with the alpha(1S) (Cav1.1) II-II V (2006) A new role for T-type channels in fast ‘low-threshold’ loop. Journal of Biological Chemistry 277: 6530–6535. exocytosis. Cell Calcium 40: 147–154. Schneggenburger R and Neher E (2005) Presynaptic calcium and Catterall WA (2000) Structure and regulation of voltage-gated control of vesicle fusion. Current Opinion in Neurobiology 15: þ Ca2 channels. Annual Review of Cell and Developmental 266–274. Biology 16: 521–555. Snutch TP (2005) Targeting chronic and neuropathic pain: Catterall WA, Perez-Reyes E, Snutch TP, and Striessnig J (2005) The N-type calcium channel comes of age. NeuroRx 2: 662–670.

International Union of Pharmacology. XLVIII: Nomenclature Talavera K and Nilius B (2006) Biophysics and structure–function þ relationships of T-type Ca2 channels. Cell Calcium 40: and structure–function relationships of voltage-gated calcium 97–114. channels. Pharmacological Reviews 57: 411–425. Chemin J, Traboulsie A, and Lory P (2006) Molecular pathways Terlau H and Olivera B (2004) Conus venoms: A rich source of underlying the modulation of T-type calcium channels by neu- novel ion channel-targeted peptides. Physiological Reviews 84: rotransmitters and hormones. Cell Calium 40: 121–134. 41–68. Davies A, Hendrich J, Tran Van Minh A, Wratten J, Douglas L, and Van Petegem F, Clark KA, Chatelain FC, and Minor DL (2004)

Dolphin AC (2007) Functional biology of the a2d subunits of Structure of a complex between a voltage-gated calcium channel voltage-gated calcium channels. Trends in Pharmacological beta-subunit and an alpha-subunit domain. Nature 429: Sciences 28: 220–228. 671–675.

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