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voltage-gated calcium channels as well as the sodium leak channel NALCN. Protein accession ... specific T-type channel blockers,7,8 which should facil-.
Advanced Review

Cav3 T-type calcium channels Adriano Senatore,1 Boris S. Zhorov2 and J. David Spafford1∗ T-type channels are unique among the voltage-gated calcium channels in their fast kinetics and low voltages of activation and inactivation, the latter two features allowing them to operate at voltages near the resting membrane potential of most neurons. T-type channels can therefore be recruited by subthreshold depolarizations, and hyperpolarizations that remove inactivation. As such, T-type channels can significantly influence how and when cells reach action potential threshold, and thus are critical regulators of excitability. T-type channels are also significantly conserved within the animal kingdom, present even in animals lacking muscles and nerves, suggesting that they evolved before or very early on during the emergence of neuronal and neuromuscular synapses. Physiologically, T-type channels are involved in multiple processes, and their contributions range from purely electrogenic roles to the activation of calcium-sensitive ion channels, signaling pathways, and other macromolecular complexes. Unfortunately, it has been difficult to prove sufficiency and necessity of T-type channels in many of these processes, in part due to inconsistencies in their suspected contributions. Furthermore, gene knockout studies have failed to show that T-type channels are essential for development or survival, as knockout animals exhibit only weak phenotypes. T-type channel roles are likely dependent on cellular context, and the three mammalian isotypes are expected to be somewhat redundant in their functionality, but have evolved from the single ancestral precursor gene in invertebrates to carry out unique functions, as evidenced by their divergent biophysical properties and protein–protein interaction motifs present within cytoplasmic regions. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. How to cite this article:

WIREs Membr Transp Signal 2012, 1:467–491. doi: 10.1002/wmts.41

INTRODUCTION

V

oltage-gated calcium channels of the Cav 3 family, or T-type calcium channels, likely evolved in animals either before or during the very early stages of nervous system evolution, and were subsequently adapted for electrical excitability and calcium signaling in different cell types including neurons, muscle and secretory cells. Although intensively studied, the lack of specific blockers has made it difficult to ascribe T-type channels with specific functions. Recent advances in imaging and electrophysiological techniques, along with the discovery of novel-blocking compounds and the ∗ Correspondence

to: [email protected]

1

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada 2

Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

Volume 1, July/August 2012

development of molecular and biological tools, have paved the way for accelerated discovery. A clearer picture is emerging, where T-type channel functions are ascribed to particular Cav 3 isotype(s), and which vary depending on the configuration of calcium-sensitive factors (i.e., calcium-sensitive signaling pathways and ionic conductances) present in cells, and also on the electrical properties of cells as determined by other ion channels, receptors, and pumps.

HISTORY OF T-TYPE CURRENT ISOLATION T-type calcium currents were first isolated in the 1970s by Susumu Hagiwara in invertebrate starfish eggs using two-electrode voltage clamp.1 ‘Channel I’ currents were evoked by small depolarizations,

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(a)

(b) Yeast CCH1

2

25

1

Drosophila Cav3 Caenorhabditis Cav3 Human Cav3.3 Human Cav3.1 Human Cav3.2

T-type

0

Membrane current, 10−7 A

[Na]out [Ca]out

Nematostella Cav3 Lymnaea Cav3

Trichoplax Cav3 Amphimedon Cav1

−60

−40

−20

Trichoplax Cav1 Nematostella Cav1

20 40 Membrane potential, mV

Human Cav1.1 Human Cav1.2 Human Cav1.3 Human Cav1.4 Lymnaea Cav1

−1

Drosophila Cav1 Trichoplax Cav2 Nematostella Cav2 Human Cav2.3 Human Cav2.1 Human Cav2.2

−2

Lymnaea Cav2 Drosophila Cav2 Trichoplax NALCN Nematostella NALCN Human NALCN Lymnaea NALCN

−3

Drosophila NALCN

Non-L-type

−80

NALCN

−100

L-type

Salpingoeca Cav1

FIGURE 1 | (a) Current–voltage plot from Hagiwara et al.,1 revealing the existence of two calcium currents in starfish eggs, ‘channel I’ currents elicited by small depolarizations, and ‘channel II’ currents requiring stronger depolarizations to reach their maximum value. (b) A phylogenetic tree showing that the most primitive calcium channels from sponge (i.e., Amphimedon) and choanoflagellates (i.e., Salpingoeca ) resemble L-type channels. Invertebrates like the snail Lymnaea stagnalis have single representatives of each of the three voltage-gated calcium channel families (LCav 1, LCav 2, and LCav 3). Interestingly, Trichoplax, simple multicellular organisms lacking nerves and muscle cells, have a full complement of voltage-gated calcium channels as well as the sodium leak channel NALCN. Protein accession numbers, listed in order as presented in the tree (from top to bottom), are P50077, 170705, AAO83843, NP_001096889, AAP79882, Q9POX4, 043497, 095180, 21513, 228755, EGD78396, 19329, 88037, Q13698, Q13936, Q01668, O60840, AAO83838, Q24270, 53006, 59997, Q15878, O00555, Q00975, AAO83841, P91645, 18273, 93026, NP_443099, AAO84496.1/AAO85435.1, and AAN77520. All protein IDs are NCBI, with the exception of Trichoplax and Nematostella (JGI Genome Portal) and Amphimedon (Amphimedon Metazome).

distinguishable as a low voltage hump in current amplitude versus test potential plots, alongside smaller ‘channel II’ currents, elicited by stronger depolarizations (Figure 1(a)). Subsequent voltage clamp studies also identified channel I currents in vertebrate preparations, including guinea-pig inferior olivary2 and thalamic neurons3 and chick sensory neurons.4 The term ‘T-type channel’ was coined on the basis of the transient (rapid) kinetics and tiny unitary conductance of channel I type currents when barium ions were the charge carrier.5 T-type or lowvoltage-activated (LVA) calcium currents could be separated from high-voltage-activated (HVA: channel II) currents such as ‘L-type’, which have longlasting single-channel openings with large unitary barium currents. L-types are easily distinguished by their sensitivity to antihypertensive, calcium channel blockers, such as the 1, 4-dihydropyridines, from other ‘non-L-type’ currents in the HVA class. ‘Non-L-types’ are responsible for transmitter release of synaptic vesicles associated with presynaptic terminals, and can be clarified further as ‘N-type’ associated with 468

neurons of mostly intermediate unitary conductance, blockable with specific ω-conotoxins isolated from cone snail venom, and ‘P- type’ currents from cerebellar Purkinje neurons or ‘Q-type’ currents in cerebellar granular neurons blockable with ‘P/Q-type’ spider venom toxin AgaIV. An ‘R-type’ current, originally isolated in cerebellar granular cells, is often a residual non-L-type current, not distinguished as N-type or P/Q-type (Figure 1(b)). Investigation into the roles of T-type channels in native cells has been limited by the absence of selective blockers as discriminatory as those used for L- and non-Ltype calcium channels. Without a selective blocker, T-type currents are not easily separated from Ltype or non-L-type currents, which often produce more robust calcium entry into the same cells. T-type currents can also be occluded by other subthreshold currents open at rest, such as persistent sodium and leak conductance currents, or currents through Kv 4 (A-type) potassium and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels which can be activated by hyperpolarization.

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WIREs Membrane Transport and Signaling

Cav 3 T-type calcium channels

TABLE 1 Inventory of Known Voltage-Gated Calcium Channel Subunits, Sodium Channels, and NALCN Homologs from Various Organisms Species Vertebrates

Snail/fly Cnidarians Placozoan Sponge Choanoflagellate Yeast

Cav 1

Cav β

Cav 2

Cav 3

NALCN

Cav 1.1 Cav 1.2 Cav 1.3 Cav 1.4 Cav 1 Cav 1 Cav 1 Cav 1 Cav 1 Calcium channel

Cav β1 Cav β2 Cav β3 Cav β4 Cav β Cav β Cav β Cav β Cav β

Cav 2.1 Cav 2.2 Cav 2.3

Cav 3.1 Cav 3.2 Cav 3.3

NALCN

10 Nav

Cav 2 Cav 2 Cav 2

Cav 3 Cav 3 Cav 3

NALCN NALCN NALCN NALCN

2 Nav Nav Nav

T-type channel functions and their contributions to cellular excitability have mostly been inferred by the characteristics of cloned cDNAs in heterologous expression systems such as HEK-293 cells and Xenopus oocytes.6 Newly discovered compounds such as the pyridyl amide TTA-P2 (3,5-dichloro-N-[1(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoropiperidin-4-ylmethyl]-benzamide) are emerging as specific T-type channel blockers,7,8 which should facilitate physiological characterization in situ and in vivo. In addition, the dynamic clamp technique, which can add or subtract ionic currents during electrophysiological recording in real time, has proven useful to reveal that even subtle changes in T-type channel conductance can have profound influences on neuronal excitability.9

GENERAL FEATURES Genomics and Evolution The mammalian genome has 10 calcium channel genes corresponding to recorded calcium channel currents, distinguished on the basis of sequence similarity between α1 pore-forming subunits.10 These consist of four L-type (Cav 1.1–Cav 1.4), three nonL-type (Cav 2.1 or P/Q-type, Cav 2.2 or N-type, and Cav 2.3 or R-type), and three T-type channels (Cav 3.1, Cav 3.2, and Cav 3.3; Figure 1(b)). The most ancient calcium channel type is likely an L-type homolog, present in single-celled eukaryotes such as choanoflagellates.11 Interestingly, even the most primitive multicellular organisms, which lack organs, internal structures, or an identifiable nervous system (i.e., placozoan Trichoplax adhaerens), have both Cav 2 and Cav 3 channels (Figure 1(b), Table 1) in addition to many proteins critical for synaptic transmission.12 Conceivably, these proteins were adapted by more complex animals to produce nerves Volume 1, July/August 2012

Na

Nav

and muscle tissue capable of elaborate electrochemical cell-to-cell signaling.13 Invertebrates mostly have single representatives of all three classes of calcium channels14–16 (e.g., snail LCav 1, LCav 2, and LCav 3 genes), which evolved into a total of 10 calcium channels as a result of genome duplications, taking on more specialized functions within the vertebrate lineage (Figure 1(b)). The least number of changes in T-type channel protein sequences from the single invertebrate T-type LCav 3 is Cav 3.1/CACNA1G followed closely by Cav 3.2/CACNA1H (Figure 1(b)). Remarkable is the relative invariance in the gating properties and voltage sensitivities between Cav 3.1, Cav 3.2 and the structurally divergent snail LCav 3 channel,16 underscoring a functional constraint on LVA calcium currents across the animal kingdom. Cav 3.3/CACNA1I is more distantly related to LCav 3 than the other vertebrate T-type channels (Figure 1(b)), and also has distinct electrophysiological properties from the other two vertebrate isotypes.

Structural Determinants of Voltage Sensitivity and Ion Selectivity Crystallization has eluded the large heterotetrameric eukaryotic channels, but approximates of coordinates can be inferred by homology modeling onto resolved X-ray structures of homotetrameric potassium17,18 and sodium channels.19 Voltage-gated calcium channels like their closest relatives, voltage-gated sodium channels and sodium leak channel NALCN, are formed by the folding together of a single polypeptide chain of four homologous repeats (domains I–IV; Figure 2(a)). Like subunits of voltagegated potassium channels, each repeat domain of a calcium channel contains six transmembrane helices (S1–S6) and membrane reentering pore loops (Ploops; Figure 2(a)). A voltage-sensor domain contains

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DI

(a)

DII

DIII

DIV

Pore loops

S4 helices figure B

NT

L45 Exons 25c and 26

Gating brake figure D

(b)

Exon 8b

DI

(c)

DII

Human Cav3.1 Lymnaea Cav3 Trichoplax Cav3 Human Cav1.2 Human Nav1.1 Human NALCN

DI

DII

DIII

DIV

Human Cav3.1 Lymnaea Cav3 Trichoplax Cav3 Human Cav1.2 Human Nav1.1 Human NALCN

DIII

DIV

Human Cav3.1 Lymnaea Cav3 Trichoplax Cav3 Human Cav1.2 Human Nav1.1 Human NALCN

(d)

CT

Human Cav3.1 Lymnaea Cav3 Trichoplax Cav3 Human Cav1.2 Human Nav1.1 Human NALCN

IS6

Helix 1

Loop

Helix 2

Human Cav3.1 Human Cav3.2 Human Cav3.3 Lymnaea Drosphila Caenorhabditis Nematostella Trichoplax Consensus

FIGURE 2 | (a) Schematic illustrating the predicted topology for voltage-gated sodium and calcium channels. One large polypeptide forms four homologous domains (DI–DIV), which are separated by large intracellular linkers. Each domain consists of six transmembrane helices, termed segments 1–6 (S1–S6). S1–S4 make up the voltage-sensor domain, with S4 (red helix) harboring positively charged residues critical for voltage sensitivity. S5, S6, and the P-loop from each domain come together to form the pore of the channel. Alternative splice sites conserved between vertebrate T-type channels and the snail LCav 3 channel in the I–II and III–IV cytoplasmic linkers are illustrated. (b) Amino acid sequences of the S4 α helices from various ion channels, revealing the presence of multiple highly conserved positively charged residues (in red). (c) Selectivity filter amino acid sequences from various ion channels including sodium and calcium. For T-type calcium channels, two glutamate (E) residues from domains I and II and two aspartate residues (D) from domains III and IV join together to form the filter. High-voltage activated calcium channels use four glutamates, while sodium channels have aspartate (D), glutamate (E), lysine (K), and alanine (A) in domains I–IV, respectively. Highly conserved tryptophan residues (W; in yellow) are in positions p53 from each domain, and calcium channels also have a highly conserved aspartate in domain II position p51, proposed to function as a molecular beacon to attract incoming calcium ions into the pore (see text). (d) Amino acid sequence of the gating brake from various T-type channels, including from one of the most primitive multicellular organism Trichoplax. Predicted helix-loop-helix structures are illustrated with two black boxes (helices) flanking the predicted loops. Amino acids are colored based on the physical properties (i.e., red is positively charged, green is negatively charged, yellow is hydrophobic, blue is hydrophilic). Secondary structures were predicted using the PSIPRED Protein Structure Prediction Server (http://bioinf.cs.ucl.ac.uk/psipred/).

four helices (S1–S4) with canonical positive charges alternating every third amino acid along a mobile α helix of S4 that moves out of the membrane in response to voltage changes (Figures 2(a),(b) and 3(a)). Each of the four voltage-sensing domains is tethered by a 470

linker helix (L45) to the ion-conducting pore domain (Figures 2(a) and 3(a)). The latter is made up of four outer S5 helices, four P-loops, and four pore-lining S6 helices. Four key acidic residues (glutamate or aspartate), located within each of the four P-turns,

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WIREs Membrane Transport and Signaling

Cav 3 T-type calcium channels

(a)

P-turn

Voltage-sensor domain

Pore domain

(b)

(c) Z

E3p50

E2p50

Ca2+ Ca2+

E4p50

E1p50

Z=0

FIGURE 3 | (a) Illustration of how P-loops between segments 5 and 6 project into the pore to form the selectivity filter. Shown only are domain I and II. Segments 1–4 form the voltage-sensor domain, tethered the pore domain via the L34 helix. Acidic amino acids critical for calcium selectivity are thought to lie within the P-turn, and four of these project into the pore to create the selectivity filter. (b) Illustration looking from the extracellular side into the pore of a voltage-gated L-type calcium channel, showing the predicted coordination of two calcium ions in the pore by the four glutamates present in the P-loops of each domain. (c) Illustration of the domain II p51 aspartate (D2p51), projecting into the pore above the p50 selectivity filter glutamate. (B and C were reprinted with permission from Ref 22. Copyright 2010 Springer)

contribute to the selectivity filter and are assigned relative numbers p5020 (Figure 3(b)). Glutamates in positions p50 form an ‘EEEE’ ring of flexible carboxylate side chains presumed to project into the pore and chelate calcium ions in Cav 1 and Cav 2 channels. Introducing at least one positively charged lysine at this critical position lowers calcium selectivity to a configuration that more resembles voltagegated sodium channels (DEKA) and NALCN sodium leak conductance channels21 (EEKE; Figure 2(c)). The ubiquitous signature sequence of the Cav 3 channel selectivity filter has two aspartate replacements of the glutamate residues compared to Cav 1 and Cav 2 (i.e., EEDD vs EEEE). Shortened carbon side chains in repeats III and IV aspartates are expected to bind calcium ions less effectively, allowing for the faster kinetics found for T-type currents at the expense of reduced calcium selectivity over monovalent cations (but see Ref 21). A reduced selectivity of Volume 1, July/August 2012

T-types is evident as an approximately 20 mV less positive reversal potential compared to Cav 1 and Cav 2 channels, and further confirmed in experiments looking at the different contributions of monovalent cations to inward and outward currents.21 Besides ubiquitous, acidic-selectivity filter residues in positions p50, the P-turns of calcium channels contain exceptionally conserved tryptophans in positions p5223 (Figure 2(c)). Interrepeat H-bonds between side chains of p52 tryptophans and side chains of p48 threonines appear to stabilize the outer pore structure in the proteobacterium NAv Ab sodium channel.19 Similar interrepeat H-bonds are expected to stabilize the outer pore structure in NALCN and calcium channels. A ubiquitous aspartate residue (D2P51 ) of calcium channels is in close proximity and wellpositioned to serve as a ‘molecular beacon’ to attract incoming calcium ions to the outer pore (Figures 2(c) and 3(c)).

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Channel Gating and the Gating Brake Voltage-gated channels transition between open (activated), closed (resting), and inactivated (refractory) states by means of voltage-sensitive gates. T-type channels are distinguished by activating at much lower voltages, allowing them to function close to resting membrane potential, while HVA channels require stronger depolarizations to become activated. A prominent mechanism for T-type channel gating occurs within the pore, as inactivation rates appear to mirror changes in activation rates, and these are altered when the EEDD locus is disrupted in the selectivity filter.21 A second inactivation gate, conserved in all voltage-gated channels, is formed by an ‘inverted tepee’ bundle of four S6 helices that occludes ion passage through the aqueous pore cavity at the cytoplasmic end of all voltage-gated channels.21,24–26 The helical structure extends from the S6 helix at the end of domain I into the most proximal region of the cytoplasmic I–II linker, forming an extended and rigid platform for interaction with accessory β subunits at the alpha interaction domain (AID) in Cav 1 and Cav 2 calcium channels. Coupling of different β subunits to the AID site differentially modulates the kinetics of Cav 1 and Cav 2, creating functional diversity.27,28 The proximal I–II linker of T-type channels is also involved in regulation of channel kinetics, but it does not involve association with β subunits. T-type channels may have lost this interaction from an ancestral precursor, as L-type channels and the β subunit, but not T-type channels, appear to be present in noneumetazoan genomes (Table 1). Instead, a highly conserved helix-loop-helix ‘gating brake’ structure replaces the β subunit interaction platform in all T-types,24 starting with those within the most primitive multicellular eukaryotes (i.e., placozoa; Figure 2(a),(d)). The second helix of this rigid structure is proposed to stabilize the channels in the closed state at hyperpolarized potentials by interacting with S6 segments of the intracellular inactivation gate.24 When deleted, the trademark features of T-type channels are enhanced even further, where mutant channels open more readily at even lower membrane voltages (i.e. hyperpolarizing shifts between −10 and −15 mV) and have even faster, more transient kinetics than normal T-type channels.29

ELECTROPHYSIOLOGICAL PROPERTIES AND CONTRIBUTIONS TO CELLULAR EXCITABILITY Calcium Versus Charge Transfer T-type and other voltage-gated calcium channels, by virtue of their ion selectivity, are at least in 472

principle able to modify other ionic conductances that are calcium-sensitive, such as potassium30 and chloride.31,32 In some instances, the calcium ion is of little consequence, exemplified in thalamocortical neurons where the contribution of T-type channels to burst firing seems to mostly depend on charge transfer across the cell membrane.9 In other cases, functional coupling between calcium channels and other calciumsensitive channels occurs, and is dependent on proximity between the proteins due to strong endogenous buffering and sequestration of cytosolic calcium.33 Indeed, T-type channels have been shown to physically interact with A-type potassium channels34 and closely associate with small conductance potassium (SK) channels,35 modulating their activity through calcium influx. T-type channels have also been implicated in calcium-induced calcium release (CICR) from internal stores by activating calcium-permeable ryanodine and inositol 1,4,5-triphosphate (IP3 ) receptors.36–39 The influence that T-type channels have in particular cells therefore depends not only on their intrinsic ion-conducting properties but also on their functional association with other calcium-sensitive conductances (see below).

High-Frequency Firing Cloning and heterologous expression of T-type channels has allowed for a detailed evaluation of their biophysical properties, although these experiments usually take place at room temperature where channel kinetics may not reflect the body temperature of mammals at around 37o C.6,40 Cav 3.1 and Cav 3.2 have strikingly similar characteristics with rapid and transient activation and inactivation kinetics and near identical voltage thresholds of activation in vitro.41 These fast kinetics lead to attenuation of peak calcium currents due to accumulated inactivation during high-frequency burst firing (>10 Hz; Figure 4(a)), exerting a temporal limit to the contribution of these two channel subtypes to prolonged excitability9,41 (Figure 4(b)). Cav 3.1 has marginally faster kinetics and a faster rate of deactivation (transition from the activated to the closed state) and deinactivation (transition from the refractory, inactivated state to the closed state) than Cav 3.2, which together account for the faster attenuation of Cav 3.1-mediated currents in a burst when compared to Cav 3.2. The more divergent Cav 3.3 has slower activation and inactivation kinetics, faster deactivation, and more depolarized voltage dependencies for activation and inactivation than the other two vertebrate subtypes. During a rapid burst, Cav 3.3 channels first facilitate, with a slight increase in peak current amplitude followed by fairly

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Cav 3 T-type calcium channels

(a)

(b) 0 mV

−70 mV

α1G

α1G

50 mV 200 ms

α1i

α1H

60 ms

(c)

α1H

Burst firing

10 mV

Regular firing

α1i

40 ms

−55 mV −75 mV

(d)

(e)

Control

100 μM nickel

G/Gmax or I/Imax

1.0 0.8 0.6 0.4

Activation (G/Gmax)

0.2

Inactivation (I/Imax)

0.0 −80

−60 −40 Voltage (mV)

−20

0

FIGURE 4 | (a) High-frequency action potentials (top trace) applied to the three vertebrate T-type channels in transfected cells (Cav 3.1, α1G; Cav 3.2, α1H; and Cav 3.3, α1I) reveal significant differences in the inactivation of their inward currents during a burst. Cav 3.1, having the fastest activation and inactivation kinetics, inactivates quickly, while Cav 3.3, which has the slowest kinetics, first facilitates then maintains relatively stable inward current amplitudes. (b) Changing T-type channel parameters to match the divergent biophysical properties of Cav 3.1, Cav 3.2, and Cav 3.3 in model thalamocortical neurons leads to significant differences in burst firing, where Cav 3.1 produces less bursting than Cav 3.2 and considerably less Cav 3.3. (A and B were reprinted with permission from Ref 42. Copyright 2002) (c) Illustration of two modes of firing for thalamic relay neurons: tonic and low-threshold calcium potentials with burst firing. Regular or tonic firing occurs from depolarized membrane potentials near −55 mV (left). Hyperpolarization to −75 mV results in postinhibitory rebound excitation generating low-threshold calcium potentials crowned by bursts of high-frequency sodium action potentials (right). (Reprinted with permission from Ref 44. Copyright 1998) (d) Reversed role for T-types in generating low-threshold calcium potentials and burst firing. Tonic firing mode (top trace) is disrupted by nickel block of T-type channels in midbrain dopamine neurons, resulting in a firing mode (bottom trace) reminiscent of low-threshold calcium potentials of the thalamus. (Reprinted with permission from Ref 35. Copyright 2002 Society for Neuroscience) (e) Overlapping steady state activation (G/Gmax) and inactivation (I/Imax) curves for the snail T-type channel reveal a voltage range near resting membrane potential where subsets of channels are available for activation and not completely inactivated, permitting a constant flux of calcium into the cell in so-called window currents. (Reprinted with permission from Ref 16. Copyright 2010 American Society for Biochemistry and Molecular Biology).

consistent peaks with little attenuation42 (Figure 4(a)). Facilitation of Cav 3.3 currents is ascribed to its slower kinetics and depolarized voltage dependencies, while, for unknown reasons Cav 3.2 currents also facilitate at very high frequencies,41 suggesting that facilitation may be a more universal property of T-type channels. Notably, the kinetics of Cav 3.3 significantly vary depending on the expression system43 (i.e., HEK vs neuroblastoma cell line NG108-15 vs Xenopus oocytes), suggesting that in some circumstances the Volume 1, July/August 2012

channel might exhibit activation and inactivation kinetics more akin to Cav 3.1 and Cav 3.2.

Low Threshold Calcium Potentials Depolarizing T-type channel currents are critical for the formation of low-threshold calcium potentials (LTCPs; also referred to as low-threshold spikes) that trigger rhythmic burst firing classically associated with thalamic neurons during nonrapid eye movement (NREM) sleep.45 LTCPs also occur in other areas of

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the brain such as the cerebellum and neocortex.9,40 In these cells, strong hyperpolarizations mobilize an active T-type channel pool by relieving inactivation, where T-type currents promote the crowning of LTCPs with high-frequency, sodium channeldependent spikes sitting on depolarized plateaus contributed by T-type channels and higher threshold voltage-gated calcium channels (Figure 4(c)). Critical determinants for the trigger of high-frequency burst firing in LTCPs are the voltage sensitivities of resident T-type channels, where subtle changes in activation and inactivation can have profound influences.9 An interesting and somewhat contradictory circumstance occurs in substantia nigra dopaminergic neurons (DA neurons), which like thalamic neurons possess tonic and burst firing modes. Unlike in the thalamus, where activated T-type channels promote LTCPs and burst firing, activated DA neuron T-type channels inhibit burst firing and rather maintain tonic firing precision.35 Pharmacological block of T-type channels leads to burst firing in DA neurons, reminiscent of LTCPs in the thalamus (albeit with a slower time course; Figure 4(d)). This apparent role reversal is accounted for by the functional coupling between Cav 3 channels and calcium-sensitive small conductance potassium channels (SK). Here, T-type channels enhance SK channel hyperpolarizing currents and in turn promote inactivation of SK channels. A reduced SK channel activity limits the afterhyperpolarization required for generating postinhibitory rebound excitation and burst firing.

neurons35 leading to stronger afterhyperpolarizations and use-dependent inactivation mirroring that of the calcium channels themselves. The capacity of T-type channels to modulate hyperpolarizing currents is also evident in granule cells in the cerebellum, where Cav 3.2 and Cav 3.3 directly couple to A-type (Kv 4) potassium channels causing a 10-mV depolarizing shift in the steady-state inactivation of Kv 4, thereby increasing the available pool of potassium channels34 and hence hyperpolarization. The role of T-type channels in detecting coincident signals and synaptic integration is suggested by their postsynaptic localization50–53 and their association with mGluR1 receptors in Purkinje neuron dendrites in the cerebellum54 and mitral cells of the olfactory bulb,52 which can potentiate T-type currents to amplify their effects. While it is clear that postsynaptic T-type channels enhance excitability in mitral cells, there is some debate over whether Ttype calcium spikes propagate to the soma of Purkinje cells in vivo (reviewed in Ref 55). Calcium-sensitive large conductance (BK) potassium channels activated by P-type calcium channel currents are thought to attenuate calcium spikes, so synaptically activated Ttype channels are expected to contribute to somatic excitability within the short time frame after excitatory input but before BK channel activation.56 Additional, nonelectrogenic roles for dendritic T-type channels are also proposed in Purkinje and other neurons including contributions to synaptic plasticity, secretion of retrograde signals55,57 (e.g., endocannabinoids and glutamate; see section on Exocytosis), and calcium-dependent restructuring of dendrites during development.58,59

Coincidence Detection and Synaptic Integration

Window Currents

Distinctive biophysical properties of T-type channels render them uniquely sensitive to changes in membrane potential near resting levels, allowing them to effectively translate incoming synaptic signals (inhibitory or excitatory) to the soma. T-type channels can be recruited by either excitatory post-synaptic potentials (EPSPs) under hyperpolarized conditions or by inhibitory post-synaptic potentials (IPSPs) that relieve inactivation under depolarized conditions.9 Focal excitatory stimulation along distal dendrites is amplified by activated T-type channels in nucleus reticularis thalamic (NRT) neurons,46,47 and similarly in hippocampal pyramidal neurons48 and neocortical neurons.49 The contribution of T-type channels to excitability of the cell soma is more complex in some cell types. As mentioned above, T-type channels can directly activate SK potassium channels in DA 474

A trademark of T-type channels is a small, continuous stream of calcium influx known as a window current through channels open at rest caused by overlapping activation and availability curves7,60,61 (Figure 4(e)). Window currents peak at resting membrane potentials, and involve only a few leaky, open channels (∼1% of the population), while the majority of T-type channels are either inactivated or closed. This trickle of calcium influx is enough to raise the resting membrane potential and promote the slow oscillatory ‘up-state mode’ of thalamic neurons that fluctuate between bistable membrane potentials.7,41,45 Window currents are also believed to be an effective means of regulating calcium-sensitive processes in cells that do not undergo sharp changes in membrane voltage, such as vascular endothelial cells, cells undergoing differentiation, proliferating cells, and cancer cells.59,62–65

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ROLES IN DEVELOPMENT, PROLIFERATION, AND DISEASE Lessons from Knockout Animals Targeted gene knockout (KO) and loss of function studies of Cav 3 genes in mammals and nematodes have certainly validated their roles in normal and abnormal brain and heart function,66–76 but the resulting weak phenotypes suggest that strong compensatory mechanisms are at play. Interestingly, neurons appear able to respond to their environment by modifying their intrinsic electrical properties in order to tune their activity to that of their local network.77 Long-term compensatory changes in neurons and other cell types likely occur through feedback to the nucleus and alterations in the transcriptome, where the consequences of absent or nonfunctional proteins might be ‘sensed’ by the cell to execute transcriptional changes. Indeed, both intrinsic78 and extrinsic79 factors can significantly influence the expression of ion channels. Loss of function of CCA-1, the only T-type channel in nematode, produces neurons with unstable resting membrane potentials that spontaneously depolarize to reach the activation range of L-type channels, a depolarizing process normally attributable to functional CCA1.69 Vertebrates have three T-type channel genes, which often exhibit overlapping expression patterns and similar biophysical properties which might facilitate compensation in single or even double KO animals.

Expression of T-Type Channels During Development and Disease T-type channels are tightly regulated in their expression both temporally and spatially during embryonic development.80,81 While robustly expressed in the embryo (e.g., skeletal muscle, neurons, heart, endocrine glands), they are more highly selective in their expression in adult tissues.59,82 Cav 3.1 and Cav 3.2 are abundant in embryonic and neonate rodent hearts but are significantly diminished in adults.59,83,84 Loss of LVA calcium currents has also been documented in developing neurons,85–88 and other cell types (e.g., chromaffin cells89 ), although in a subset of neurons Cav 3 channel currents and proteins are developmentally upregulated.59,90–92 A number of splice sites of T-type channels also undergo differential splicing during development, signifying that there are genetic program switches and role changes for T-types from the embryo to the adult animal.83,93–95 How T-type channels contribute to development and disease is not fully understood. At a glance, Volume 1, July/August 2012

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these processes are multifactorial, context-specific, and subject to redundancy. Interesting is that embryonic-like, high-level expression65,96–99 and splicing patterns83,100 of Cav 3 channels reemerge during various diseased states and during stress. This reexpression suggests that in some cases, T-type channels are rerecruited to fulfil roles similar to those manifested during ontogenesis of the animal.

Oxygen-Sensing in Chromaffin Cells Highly expressed Cav 3.2 T-type channels are thought to contribute to exocytotic catecholamine secretion,101,102 and augmented secretion during acute hypoxia89 in embryonic adrenal medulla chromaffin cells. A drop in Cav 3.2 expression is coincidentally associated with a loss of the O2 -sensing role of chromaffin cells in adults after developmental innervation by splanchnic nerves. Exposure to chronic hypoxia or denervation of the adrenal medulla can restore Cav 3.2 channel expression and the hypoxic response phenotype.89,101,103,104

Developing and Diseased Heart Cav 3.1 and Cav 3.2 are differentially expressed in the developing heart,81 but are mostly lost after birth to a residual function in pacemaker cells.99,105 Various hormones such as insulin-like growth factor 1, angiotensin II, endothelin 1, and 17β-estradiol recruit T-type channel isoforms to the injured and diseased myocardium.83,84,99,105–108 T-type channels are expected to contribute to calcium signaling and the proliferation and growth of both developing hearts and diseased/damaged heart cells undergoing pathogenic remodeling.96,97,105 Additional roles are also likely, such as contributions to excitability, contraction and pacemaking,39,71,84,105,109–111 secretion of hormones,112 cell fate determination,113 and control of blood vessel tone67 (see below).

Neuronal Development and Disease Neuronal T-type channels largely persist into adulthood, in contrast to most other tissues, albeit with differences in expression and splicing.59,80 T-types are implicated in proliferation and various morphological changes in developing neurons including neuritogenesis, growth, and outgrowth of dendrites and axons.59 Dendrites of developing auditory coincidence-detector neurons undergo striking, synaptically mediated and calcium-dependent morphological changes, where dendritic T-type channels significantly influence postsynaptic excitability and calcium influx.58 Migrating axons of chick embryonic motor neurons utilize Cav 3

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channels to initiate highly controlled calcium oscillations that are critical for the expression of guidance molecules.114 Nonproliferative and nonmorphogenic roles are also proposed, such as contributing to the maturation of ionic conductances.59 L-type and nonL-type calcium channel expression is impaired with drug block or antisense knockdown of Cav 3.2 in differentiating neuroblastoma cell line NG108-15,78,115 presumably by disrupting T-type-dependent activation of a Src kinase pathway.116 In contrast, normal L-type channel currents are found in the atrioventricular node of the hearts of Cav 3.1 KO mice,71 but cardiac-specific overexpression of Cav 3.1 significantly changes the expression of various calcium-handling proteins including L-type channels, sarco/endoplasmic reticulum calcium ATPase (SERCA), and the neuronal calcium exchanger.111 T-type channels are also implicated in several diseases of the nervous system, most notably epilepsy.117,118 Thalamocortical and NRT neurons in the thalamus rely heavily on T-type channels for modulating their excitability, and alterations in Ttype channel function can have serious consequences.9 Mice lacking Cav 3.1 are unable to elicit thalamocortical LTCPs and burst firing in response to hyperpolarization, and these animals are resistant to druginduced absence seizures.66 Overexpression of Cav 3.1 in transgenic mice induces thalamocortical spike-wave oscillations characteristic of pure absence epilepsy,100 and several animal models of absence epilepsy show increases in T-type channel current density.119 Genetic screening aimed at characterizing abnormalities in putative epileptogenic genes in individuals stricken with childhood absence epilepsy revealed that a small subset of them harbored mutations in Cav 3.2.120,121 Subsequent electrophysiological studies showed that most identified missense mutations affected either or both biophysical properties and the current density of Cav 3.2 in heterologous expression systems.122–124 LTCPs of the thalamus are also important for stabilizing NREM sleep, where T-type channel activity helps block sensory information from stimulating the cortex. Not surprisingly, KO and focal deletion of thalamic Cav 3.1 produces mice with significant sleep disturbances and abnormal sleep.68,70,125 Other nervous system ‘channelopathies’ believed to arise from T-type mutation or misregulation include autism spectrum disorder,74,76,126,127 essential tremor,74,76,127 and hyperalgesia mediated by both peripheral72,128–132 and central133,134 neurons (see below).

Proliferation and Cancer Both the cell cycle and apoptosis are highly calciumdependent, and alterations in calcium handling can 476

have profound consequences.65,135 T-type channel current rises in G1 and S phases and disappears or decreases in other phases of the cell cycle in cultured smooth muscle cells, and T-types are implicated in proliferation and promoting cell cycle progression in both healthy and cancerous cells.65,136,137 Drug block or antisense-mediated silencing of T-type channels reduces cell proliferation, and cancer cell metastasis.59,65,98,138,139 T-type channel expression is frequently upregulated in cancerous cells;59,65,98 however, whether their contribution is indirect (i.e., mediated through changes in excitability, modulation of other ionic conductances, CICR, or secretion of autocrine growth factors),59,98,140 or direct (i.e., by coupling to and activating specific calcium-sensitive signaling pathways), has yet to be resolved. Heterologous overexpression of T-type channels can promote proliferation,98 but not in all cells,60 suggesting that appropriate calcium signaling configurations are required in the cytosol to couple T-type channels to proliferation.

INTRACELLULAR PATHWAYS MODULATED BY T-TYPE CALCIUM CHANNELS Rises in intracellular calcium through T-type calcium channels are known to activate a number of cellular pathways, namely extracellular signalregulated kinase (ERK),141–143 nitrous oxide synthase397 (NOS-3), and calcineurin/NFAT.96 Both Cav 3.1 and Cav 3.2 are implicated in ERK signaling in a number of cells including HEK-293,141 osteocytes,142 and proliferating cancer cells.143 The ERK pathway also feeds back to modulate T-channels at both the protein144–146 and transcript147 levels. The molecular mechanisms linking ERK pathway activation and Ttype channels are presently unclear, and close coupling between Cav 3 channels and calcium-sensitive ERK pathway members has not yet been reported. Interestingly, scaffolding protein RanBPM148 was recently found to interact with the I–II linker of Cav 3.1 and modulate its surface expression,149 and RanBPM is directly implicated in ERK pathway activation.150 Both Cav 3.1 and Cav 3.2 are pathologically reexpressed in mouse models of cardiac hypertrophy,84,99,105 where they directly interact with calcium-sensitive NOS-3. The channels appear to have opposing functions, however, where Cav 3.1 antagonizes the hypertrophic phenotype by activating NOS-3 and inhibiting the prohypertrophic calcineurin/NFAT pathway,97,151 while Cav 3.2 agonizes hypertrophy by directly activating calcineurin/NFAT.96 Interestingly,

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nitrous oxide (N2 O) produced by activated NOS enzymes can effectively attenuate Cav 3.2 and less so Cav 3.1 currents,152,153 and the involvement of these two channel subtypes in cardiac hypertrophy might be orchestrated by a complex interplay between the channels themselves and their associated downstream effectors.

OTHER PHYSIOLOGICAL ROLES FOR T-TYPE CHANNELS Exocytosis T-type channels are implicated in calcium-dependent fusion of synaptic and large-dense core vesicles in neurons and secretory cells,103 and in nonexcitable cells such as pulmonary vascular endothelial cells62,63 and cancer cells.59,140 Although their contribution is often indirect and limited to membrane depolarization and activation of HVA calcium channels which mediate vesicle fusion,69,154 some reports suggest that T-type channels are important for low-threshold exocytosis, as occurs during spontaneous release of synaptic vesicles at rest in peripheral neurons (i.e., mini EPSPs),155 and during graded synaptic transmission at voltages below action potential threshold in both vertebrate and invertebrate neurons.57,103 T-type channels are involved in the acrosome reaction in sperm during fertilization, where they deinactivate after the membrane potential drop associated with capacitation and contribute to acrosomal exocytosis156,157 (though see Ref 158). Whether T-type-mediated exocytosis is dependent on close association of the channels with calcium-sensitive fusion proteins, as is common with N- and P/Q-type channels, or occurs via global rises in intracellular calcium remains to be clarified.103

Contraction and Smooth Muscle Tone Embryonic and postnatal rat heart myocytes have underdeveloped transverse-tubules (T-tubules), during which contraction is mostly dependent on influx of extracellular calcium, which may involve T-type channels. As myocytes mature, T-tubules progressively develop and excitation–contraction coupling driven by Cav 1.2 channels that trigger calcium release from intracellular stores prevails.84,105 This transition coincides with the loss of expression of T-type channels,108 whose abundance in embryonic myocytes suggests that they contribute to early forms of contractile activity, before T-tubule development. Interestingly, reexpression of T-types in the adult heart myocardium, either during disease or in transgenic mice, produces only slight changes in contractility but substantial Volume 1, July/August 2012

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compensatory changes in other calcium-handling channels, pumps, and exchangers.111 T-type channels are not involved in contraction of vertebrate skeletal muscle cells, but are implicated in developmental myoblast fusion.59 Invertebrates might rely more heavily on T-types for contraction of both heart and muscle cells. Significant LVA calcium currents have been recorded in jellyfish muscle159 and mature snail ventricular myocytes,160 and the Caenorhabditis elegans T-type CCA-1 significantly contributes to excitation–contraction coupling and shape of muscle cell action potentials.161,162 Mibefradil, used clinically and initially marketed as an antihypertensive, T-type selective blocker, was retracted for its dangerous side effects163 and later found to block sodium, potassium, chloride, and other calcium channels with considerable affinity.164–166 Subsequent studies also revealed that the antihypertensive action of mibefradil is mostly dependent on the block of L-type channels.167,168 The most convincing evidence implicating T-type channels in the control of vascular smooth muscle tone and blood pressure comes from peripheral vasculature in which only Cav 3 channel currents can be detected, and their block significantly disrupts smooth muscle contractility.169 In vascular myocytes of the mouse heart, KO of Cav 3.2 results in a loss of N2 O-mediated relaxation,67 suggesting that under normal conditions Cav 3.2 contributes to a basal, steady-state tone that can be relieved with N2 O, a direct inhibitor of the channel.152,153,170 Vascular smooth muscle cells often have fairly depolarized membrane potentials (i.e., ∼ −40 mV171 ), where T-type channel activity would be restricted to tonic calcium influx via window currents.169 Conversely, some smooth muscle cells maintain more negative resting potentials,172 which is a prerequisite for T-type channels being involved in generating oscillating calcium waves and pacemaking, as has been proposed in various smooth muscle cells including those in the vasculature,168 the uterus,173,174 and the bladder.172

MODULATION OF FUNCTION G-Proteins, Kinases, and Lipids Cav 3.2 channels possess the greatest degree of transient modulatory capacity of the three mammalian Cav 3 subtypes.175 Regulatory pathways activated by neurotransmitters and hormones most commonly modulate T-type currents through G-protein-coupled receptors (GPCRs), such as mGluR1, lysophosphatidic acid, opioid, D1 and D2 dopamine, serotonin, M1 muscarinic, gamma-aminobutyric acid B (GABAB ), angiotensin II AT1 and AT2,

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corticotrophin-releasing factor-1 (CRF-1), neurokinin1, and chemokine CCR2 receptors.55,175,176 Ligand binding to specific GPCRs activates a complement of G protein α and βγ subunits. The α subunits modulate kinases with multiple phosphorylation substrates including the cytoplasmic linkers of Cav 3 proteins (e.g., protein kinases A, C, and G, membraneassociated protein kinase, and Rho kinase176–178 ). Phosphorylation-mediated inhibition of T-type channels is reportedly more frequent and tends to occur through a reduction in peak current, while stimulation often involves changes in biophysical parameters.175 The focal point of known target sites for GPCR signaling and phosphorylation is mostly the cytoplasmic II–III linker.175,176 Brain phosphoproteome analysis of several voltage-gated calcium channels, including Cav 3.1 and Cav 3.3, is consistent with the cytoplasmic II–III linkers and C-termini as hotbeds for phosphorylation, compared to the I–II linkers typically for sodium channels.179 GPCRs also inhibit Cav 3.2 channels exclusively by direct interaction of activated G protein β2 γ2 subunits with the II–III linker, reducing open channel probability without affecting voltage sensitivity.178 This interaction is dependent on phosphorylation of Cav 3.2 by protein kinase A, which is indicative of an overlap between G protein αs and β2 γ2 signaling.180 Activated M1 muscarinic and CRF-1 GPCRs are able to inhibit Cav 3.3 and Cav 3.2, respectively, via unknown mechanisms that do not involve activation of canonical kinase pathways.176 Also, neuromodulatory lipids (i.e., endocannabinoids and other related molecules), shown to inhibit HVA calcium channels via activation of Gprotein coupled cannabinoid receptors,181 also inhibit T-type currents, although independent of receptor activation or G proteins and likely through a direct interaction.182–186 Importantly, a significant amount of disparity is apparent in reports of GPCR modulation of T-channels, but this might be accounted for by interspecies differences, splice variability, differences in basal phosphorylation states before Gprotein activation, and cross-talk between different GPCR signaling pathways.175,178 As is the case with many other aspects of T-type channel biology, isotype specificity and cytoplasmic context seem critical for defining the output of given modulatory pathways. In addition, several GPCR-activated kinases are highly temperature-dependent, and research using mammalian cells at room temperature might underestimate kinase activity.187 Other pathways and kinases modulate T-type channels, including receptor tyrosine kinases175,176 and calmodulin-dependent protein kinase II (CaMKII), which respond to rises in intracellular calcium by 478

phosphorylating the II–III linker of the Cav 3.2 channel, shifting the activation curve toward more negative potentials.175,183 Of note, calmodulin itself might directly modulate T-type channels in some preparations.188

Alternative Splicing: Developmentally Regulated Isoforms that Control Gating T-type channels operate in a voltage range where slight changes in biophysical properties due to alternative splicing can have tremendous consequences.9 Multiple splice sites create variation in Cav 3.1,93,189,190 Cav 3.2,94,191 and Cav 3.3 channels,192 but there is one remarkable site worth noting in the cytoplasmic III–IV linker that is developmentally regulated83,93,95 and evolutionarily conserved. Exon 25c represents a retained intron in invertebrate LCav 3 and vertebrate Cav 3.1 and Cav 3.2 channels, respectively (Figure 2(a)). Exon 26, downstream of exon 25c, is an optional exon that together with 25c creates a pattern of splicing in the III–IV linker that includes a single lysine residue () or inserts of various sizes (exon 25C, 26, 25C+26) that range from 7 to 26 amino acids. Cav 3.3 contains only the single amino acid,  variant, and likely lost its diversity of isoforms in this region shared in the invertebrate and other vertebrate T-types.192  variants produce channels with slower activation and inactivation kinetics, and shifted activation and steady-state availability curves in the depolarizing direction.83,93,94,191,193 Heterologously expressed  variants of Cav 3.1 and Cav 3.2 resemble Cav 3.3, which is more widespread and abundantly distributed in the embryonic nervous system.50,194,195 The enrichment of  variants in the embryo83,93 provides T-type channels with features more akin to Cav 3.3, which contributes less calcium to a single action potential spike compared to other T-types, and is more resistant to cumulative inactivation and attenuation during high-frequency firing.9,41 While Cav 3.3 channels are not involved in the diastolic depolarization of cardiac myocytes, the  variant of Cav 3.2 is more prevalent in newborn hearts, has slower kinetics, and exhibits voltage-dependent facilitation not present in adult Cav 3.2 variants containing exon 25c.83 The embryonic, heart-specific isoforms of Cav 3.1 contain exon 26 exclusively,107 which relative to  variants possess faster activation and inactivation kinetics and hyperpolarized activation and steadystate availability curves.93,193 Of note is that alternative splicing of T-type channels occurs most often within intracellular regions,93,94,189,190,192 which would allow for effective, differential engagement with

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cytosolic factors. There are also consequences of III–IV linker isoforms in disease, as is evident in absence epilepsy. Seizure activity in rats caused by an arginine to proline substitution in exon 24 of Cav 3.2 (R1584P) only manifests when exon 25c is also present.196 The missense mutation in exon 24, together with exon 25c, produces channels with a faster recovery from inactivation, resulting in increasing current and epileptic burst activity.

Alternative Splicing: The Role of the I–II Linker in Membrane Trafficking Regulation of membrane surface expression is a probable factor in T-type channel-associated diseases, such as temporal lobe epilepsy197 and diabetic neuropathy,130 where the channels normally contribute to neuronal excitability, such as oscillatory thalamocortical rhythms during sleep.68,70,125 Strategic deletions in the distal I–II linker of T-type channels downstream of the gating brake (Figure 2(a)) and single nucleotide polymorphisms within this region, identified in patients with absence epilepsy,120,121 severely alter membrane trafficking.123,198 It is likely that the mechanisms that control surface expression in the I–II linker are different between the three Cav 3 subtypes, as equivalent deletions within the I–II linker have quantitatively different consequences, such as a modest increase (1.5-fold, Cav 3.1), a dramatic increase (3.7-fold, Cav 3.2), or a decrease (1.7-fold, Cav 3.3) in membrane surface expression.123,198 The I–II linkers of Cav 3.1, Cav 3.2, and Cav 3.3 are also highly divergent, making it likely that they have different associations with cytoplasmic and secretory pathway proteins. Specific factors that regulate T-type channel membrane expression include neuropoietic cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), which can upregulate T-type channel membrane trafficking in nodose ganglion neurons.145,146 The cytokines activate the Janusactivated kinase (JAK) pathway, leading to a transient activation of the ERK pathway to increase membrane trafficking.144–146 Interestingly, Cav 3 channels are directly implicated in ERK activation, suggesting that feed-forward mechanisms might allow T-type channels to upregulate their own surface expression. RanBP9 may also be involved, as it binds to and upregulates Cav 3.1 channel membrane expression,149 and is implicated in ERK activation.150 Downstream of cytokine-mediated ERK activation, increased T-type channel surface expression likely involves actindependent vesicular trafficking. Disruption of the actin cytoskeleton, or overexpression of a dominantnegative form of ADP-ribosylation factor 1 (important Volume 1, July/August 2012

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for vesicular trafficking and actin remodeling199 ), abrogates the cytokine-induced upregulation of membranous Cav 3.2.144 Cav 3.2 and non-L-type calcium channel Cav 2.1, but not Cav 3.1, interact with neuronal actin-binding protein Kelch-like 1, which acts as an adaptor to mediate actin-dependent trafficking to the membrane.200–202 Interestingly, the exogenous compound butyrate, a metabolic byproduct of intestinal flora, can upregulate Cav 3.2 surface expression in colonic nociceptive sensory neurons, where the channels are thought to contribute to chronic visceral pain during bouts of irritable bowel syndrome.132 Alternative splicing is a mechanism for altering the membrane surface expression of T-type calcium channels. Both invertebrate LCav 3 and human Cav 3.1 channels contain an exon within the I–II linker (exon 8), which has tandem alternative 5 donor splice sites capable of truncating the linkers by 134 and 201 amino acids, respectively203 (Figure 2(a)). The natural isoforms omitting exon 8b are abundant transcripts in both snails and mammals, and have no influence on biophysical properties, but approximately double the membrane expression in heterologous systems of both mammalian203 and invertebrate (Senatore and Spafford, unpublished) channels similar to strategic deletions within the I–II linker. It is tempting to speculate that this optional exon encodes an evolutionarily conserved regulatory factor, which is used to modulate the surface expression and/or total protein expression of Cav 3.1 and LCav 3 in their respective in vivo systems. Cav 3.3 also harbors an optional exon in the I–II linker;192 however, unlike Cav 3.1 and LCav 3, this exon significantly influences biophysical properties, and differences in surface expression have not been studied in detail,204 although deletions in this region reduce membrane expression in vitro.123,198

Transcription T-type channels appear to be subject to extensive regulation at the transcriptional level. This form of regulation is perhaps in lieu of the apparent lack of association of T-types with accessory subunits such as the β, α2 δ, and γ of HVA channels (although see Refs 205, 142, and 206), allowing for the up- or downregulation of surfaceexpressed channels without a requirement for posttranslational, multimeric subunit assembly. Various growth factors,207,208 hormones,147,209–211 and transcription factors147,212,213 have been implicated in T-type channel transcription. The human Cav 3.1 gene is under the control of two different promoters,

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which are differentially expressed during neuronal differentiation.88 In the thalamus, both promoters of Cav 3.1 are highly active due in part to the concerted action of LEF1 transcription factors and nuclearlocalized cell adhesion protein β-catenin.214 Cav 3.1 and/or Cav 3.2 transcript levels change during differentiation and development40,59,80,84,137,207 (see section on Development above), and in response to various stress conditions including pulmonary hypertension in pulmonary smooth muscle cells,208 intermittent hypoxia in secretory cells,102,104,215,216 neuropathic pain217 and cardiac hypertrophy and disease,96,97,99 as well as in cancerous cells.98 It is not clear whether the upregulation of T-type channel transcripts during stress and disease contributes to pathogenicity or serves to minimize damages. Both Cav 3.1 and Cav 3.2 become reexpressed in hypertrophic ventricular myocytes, but only Cav 3.2 is reported to contribute to the hypertrophy,96 while Cav 3.1 antagonizes it.97 Such competing functions are also apparent in cancer cells, where T-type channels are thought to promote cancer cell growth136,138,143,218 in most cases, while in others they suppress it.209 Hypermethylation of the CACNA1G gene occurs in various human cancers, suggesting it might have antitumorigenic functions.219–221 It seems likely that the recruitment of the three vertebrate T-type channel isotypes to specific cellular processes is differential and context-specific, requiring the specific association and/or activation of calcium-sensitive signaling molecules.

Nociception, Divalent Cation Block, and Oxidation/Reduction Cav 3.2 channels are highly localized at peripheral nerve endings and in cell bodies of afferent dorsal root ganglia (DRG) nociceptive neurons, where they contribute to stimulus-induced excitability and enhanced synaptic release of pronociceptive neurotransmitters (e.g., glutamate, substance P, calcitonin-gene related peptide) onto postsynaptic thalamus-projecting neurons.222–224 Gene or antisense knockdown of Cav 3.2,72,129 or intrathecal injection of T-type channel blockers,131,225 reduces the excitability of primary afferent pain fibers resulting in diminished pain perception. Neuronal T-channel current density and transcription are also upregulated in animal models of neuropathic pain and diabetic neuropathy,130,217,226 which is consistent with a role of T-type channels in peripheral pain pathways. Sensation of peripheral pain can be regulated endogenously, through the especially sensitive block 480

of Cav 3.2 channels by divalent cations (i.e., nickel, copper, and zinc227–230 ). The concentration of unchelated, free zinc is expected to be above the threshold in neurons to specifically dampen the activity of Cav 3.2 channels. [Zn2+ ], for example, is higher in the gray matter than the white matter of the brain, highest in forebrain structures including the hippocampus and neocortex, and intermediate levels of both copper and zinc are found in the thalamus.231,232 Both zinc and copper are also secreted presynaptically, where they are thought to directly modulate various targets including voltagegated ion channels.233 Zinc/cation block of Cav 3.2 channels can be reduced by applying exogenous or endogenous chelators (e.g., tricine and albumin, respectively), as well as exogenous and endogenous reducing agents (e.g., dithiothreitol or L-cysteine, respectively).234 Reducing agents are expected to alter the disulfide bridges formed in the extracellular loops of Cav 3.2 channels, thereby reducing zinc affinity for and its inhibition of Cav 3.2. One key residue reported for high-affinity zinc binding is histidine 191, where a histidine to glutamine substitution (H191Q) located in the repeat domain I extracellular S3-S4 linker abrogates the high-affinity block and concomitantly eliminates the sensitivity of Cav 3.2 channels to reducing and chelating agents.228–230 Animals with a gene KO of Cav 3.2 have attenuated perception of thermal pain upon peripheral injection of L-cysteine, where nociceptors lose their sensitivity to reducing agents and zinc chelators.234 All of these data suggest a model where the perception of peripheral pain is mediated through Cav 3.2 channels, which can be altered by dynamic fluctuations in endogenous zinc levels and reducing agents such as L-cysteine. Cav 3 channels are also crucial for central processing of sensory information including pain, where they play essential roles in the thalamus including generating LTCPs.45 LTCPs represent a distinct firing mode for thalamocortical neurons, which is thought to impede sensory information from reaching the cortex during NREM sleep (and during epileptic seizures).45,68,70 During the awake state, thalamocortical neurons transition to a tonic-firing mode, which permits relay of sensory information to cortical neurons. T-type channels are typically inactivated in this awake state, which is associated with depolarized membrane potentials and a diminishing ability of T-type channels to contribute to excitability. KO of GPCR-linked phospholipase C β4 (PLCβ4) in mice produces a significant upregulation of T-type channel activity in thalamocortical neurons, likely due to a reduction in protein kinase C

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activity that normally attenuates T-type currents175 (see above). Interestingly, disruption of PLCβ4 also produces T-type channels that inactivate less at depolarized potentials, allowing them to promote exaggerated thalamocortical burst firing and concurrently a significant reduction in pain sensitivity.134 Cav 3.2 is a major target for redox modulation in the thalamus235 and presumably for endogenous zinc-induced T-type current inhibition.227 Zinc also has significant effects on Cav 3.3 which is highly expressed in inhibitory NRT neurons that project onto and regulate thalamocortical neurons. In contrast to the inhibition of Cav 3.2, zinc promotes Cav 3.3 activity by slowing deactivation, producing currents that prolong the duration and increase the frequency of burst firing.227,236

NOVEL T-TYPE CHANNEL INHIBITORS T-type channels contribute to a broad range of cellular processes such as excitability and pacemaking, proliferation, differentiation, secretion, and smooth muscle contraction and tone, and abnormal T-type channel function is associated with disease. In addition, Cav 3 channels appear to be important for stress responses, and in some cases enhanced activity might be beneficial. Not surprisingly, T-type channels are one of the most sought after drug targets, and there are dozens of available patents for T-type channel blockers, including 15 different classes of chemical structures.237 Recently, new compounds are emerging with high selectivity for T-type channels,7,8 which should contribute significantly to the study of T-type physiology and function, but more importantly, open the door for discovery of tailored drugs that block T-type channels in an isotype-specific manner. Indeed, due to the broad and differential localization and function of the three vertebrate T-type channels, pharmacological compounds should be tailored not only to be specific for the channel proteins themselves but also for the tissues that are relevant for a given pathology.

CONCLUSION T-type LVA calcium currents were first described in the 1970s and 1980s along with other calcium currents, but their functions have largely remained enigmatic. T-type channels are often reported to have inconsistent or opposing roles, and their functions appear modular and dependent on the unique calcium-sensitive mechanisms present in cells. KO animals have failed to show that T-type channels are either necessary or sufficient for any Volume 1, July/August 2012

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overt developmental or physiological process, a conundrum that might be attributable to redundancy between the three mammalian isotypes and/or compensatory changes. In addition, the in vivo contribution of T-type channels in neurons has likely been underestimated, given that (1) T-type channels are enriched in dendrites, (2) space clamp issues in neuronal slice preparations can interfere with the accuracy of measurement of dendritic structures, and (3) dissociated neurons lose their dendritic arbor.238 With the advent of new blocking compounds, molecular tools, and new technologies, such as dynamic clamp, we are learning of dramatic distinctions in the roles that the different T-type channels have in vivo, and we are gaining an appreciation of how even slight alterations in biophysical properties arising from isotype specificity, alternative splicing, and modulation can have significant consequences. The three vertebrate Ttype channels can differentially associate with and modulate various signaling pathways and calciumsensitive ion channels, providing cells with a rich inventory for assembling T-type channels into specialized functional configurations. A recently characterized invertebrate Cav 3 channel from snail serves to reveal evolutionarily conserved features of T-type channels.16 Genome sequencing of primitive animals suggests that T-type channels first appeared alongside synaptic Cav 2 channels in multicellular animals before or during the very early stages of nerve, muscle, and sensory organ evolution, and likely formed from an L-type calcium channel ancestor in single-celled eukaryotes. Cav 2 channels specialized to the presynaptic, secreting end of axons, while Cav 3 channels occupied a dendritic localization to regulate the excitability of neurons, and then expanded to similar pacemaking roles when the cardiovascular system appeared later in bilateral organisms. T-type channels appear to have assumed roles in the excitability of primitive muscle, which may have been lost in muscle evolution with the advent of specialized, striated muscle and excitation–contraction coupling with T-tubules, and highly organized neuromuscular junction tetrads. Gene duplications of the single invertebrate T-type channel gene into the three vertebrate genes created uniquely specialized channels. Cav 3.2 acquired a greater modulatory capacity than Cav 3.1 channels, and expanded its roles outside of the brain and heart. Cav 3.1 and Cav 3.2 channels have similar fast kinetics, suited for rhythm-setting roles during highfrequency action potential bursts of short duration, whereas the slower kinetics of Cav 3.3 channels permit

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higher frequency action potential volleys with little attenuation. The promise of new pharmaceuticals for the treatment of epilepsy, pain, and hypertension has

led to an accelerated pursuit of selective blockers of T-type channels, which will further aid in discriminating different roles for T-type channels.

ACKNOWLEDGMENTS We would like to acknowledge Dr. Arnaud Monteil for his helpful suggestions, Dr. Adrienne Boone for editing the manuscript, and Owen Woody for his assistance in generating the phylogenetic tree diagram presented in Figure 1(b).

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11. Liebeskind BJ, Hillis DM, Zakon HH. Evolution of sodium channels predates the origin of nervous systems in animals. PNAS 2011, 108:9154–9159.

´ R, Yarom Y. Electrophysiology of mammalian 2. Llinas inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances. J Physiol 1981, 315:549–567.

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230. Kang HW, Moon HJ, Joo SH, Lee JH. Histidine residues in the IS3-IS4 loop are critical for nickelsensitive inhibition of the Cav 3.2 calcium channel. FEBS Lett 2007, 581:5774–5780. 231. Tarohda T, Ishida Y, Kawai K, Yamamoto M, Amano R. Regional distributions of manganese, iron, copper, and zinc in the brains of 6-hydroxydopamineinduced parkinsonian rats. Anal Bioanal Chem 2005, 383:224–234.

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