Functional impact of alternative splicing of human T-type Cav3.3 calcium channels

Low-voltage-activated T-type (Cav3) Ca2+ channels produce low-threshold spikes that trigger burst firing in many neurons. The CACNA1I gene encodes the Cav3.3 isoform, which activates and inactivates much more slowly than the other Cav3 channels. These distinctive kinetic features, along with its brain-region-specific expression, suggest that Cav3.3 channels endow neurons with the ability to generate long-lasting bursts of firing. The human CACNA1I gene contains two regions of alternative splicing: variable inclusion of exon 9 and an alternative acceptor site within exon 33, which leads to deletion of 13 amino acids (Delta33). The goal of this study is to determine the functional consequences of these variations in the full-length channel. The cDNA encoding these regions were cloned using RT-PCR from human brain, and currents were recorded by whole cell patch clamp. Introduction of the Delta33 deletion slowed the rate of channel opening. Addition of exon 9 had little effect on kinetics, whereas its addition to Delta33 channels unexpectedly slowed both activation and inactivation kinetics. Modeling of neuronal firing showed that exon 9 or Delta33 alone reduced burst firing, whereas the combination enhanced firing. The major conclusions of this study are that the intracellular regions after repeats I and IV play a role in channel gating, that their effects are interdependent, suggesting a direct interaction, and that splice variation of Cav3.3 channels provides a mechanism for fine-tuning the latency and duration of low-threshold spikes.     

Cav3 T-type channels: regulators for gating,membrane expression,and cation selectivity

Ca_v3 T-type channels are low-voltage-gated channels with rapid kinetics that are classified among the calcium-selective Ca_v1 and Ca_v2 type channels. Here, we outline the fundamental and unique regulators of T-type channels. An ubiquitous and proximally located “gating brake” works in concert with the voltage-sensor domain and S6 alpha-helical segment from domain II to set the canonical low-threshold and transient gating features of T-type channels. Gene splicing of optional exon 25c (and/or exon 26) in the short III–IV linker provides a developmental switch between modes of activity, such as activating in response to membrane depolarization, to channels requiring hyperpolarization input before being available to activate. Downstream of the gating brake in the I–II linker is a key region for regulating channel expression where alternative splicing patterns correlate with functional diversity of spike patterns, pacemaking rate (especially in the heart), stage of development, and animal size. A small but persistent window conductance depolarizes cells and boosts excitability at rest. T-type channels possess an ion selectivity that can resemble not only the calcium ion exclusive Ca_v1 and Ca_v2 channels but also the sodium ion selectivity of Na_v1 sodium channels too. Alternative splicing in the extracellular turret of domain II generates highly sodium-permeable channels, which contribute to low-threshold sodium spikes. Ca_v3 channels are more ubiquitous among multicellular animals and more widespread in tissues than the more brain centric Na_v1 sodium channels in invertebrates. Highly sodium-permeant Ca_v3 channels can functionally replace Na_v1 channels in species where they are lacking, such as in Caenorhabditis elegans.     

Different distribution of Cav3.2 and Cav3.1 transcripts encoding T-type Ca(2+) channels in the embryonic heart of mice

We investigated the distribution of T-type Ca(2+) channel mRNAs in the mouse embryonic heart. Cav3.2, but not Cav3.1, was expressed in the E8.5 embryonic heart along with cardiac progenitor markers (Nkx2.5, Tbx5, Isl-1) and contractile proteins (alpha and beta MHC). In the E10.5 heart, the distribution of Cav3.1 mRNA was confirmed in the AV-canal and overlapped with that of MinK or Tbx2. Cav3.2 mRNA was observed not only in the AV-canal but also in the outflow tract, along with MinK and Isl-1, indicating the expression of Cav3.2 in the secondary heart field. Thus, Cav3.2 may contribute to the development of the outflow tract from the secondary heart field in the embryonic heart, whereas Cav3.1 may be involved in the development of the cardiac conduction-system together with Cav3.2.     

The many faces of T-type calcium channels

Since the discovery of low-voltage-activated T-type calcium channels in sensory neurons and the initial characterization of their physiological function mainly in inferior olive and thalamic neurons, studies on neuronal T-type currents have predominantly focused on the generation of low-threshold spike (and associated action potential burst firing) which is strictly conditioned by a preceding hyperpolarization. This T-type current mediated activity has become an archetype of the function of these channels, constraining our view of the potential physiological and pathological roles that they may play in controlling the excitability of single cells and neural networks. However, greatly helped by the recent availability of the first potent and selective antagonists for this class of calcium channels, novel T-type current functions are rapidly being uncovered, including their surprising involvement in neuronal excitability at depolarized membrane potentials and their complex control of dendritic integration and neurotransmitter release. These and other data summarized in this short review clearly indicate a much wider physiological involvement of T-type channels in neuronal activity than previously expected.     

Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+

Aim: We have investigated the influence of Ca²⁺ ions on the basic biophysical properties of T‐type calcium channels. Methods: The Ca_v3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca²⁺ or Na⁺ ions were used as charge carriers. The intracellular Ca²⁺ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 μm Ca²⁺ into the non‐buffered intracellular solution. Various combinations of extra‐ and intracellular solutions yielded high, intermediate or low intracellular Ca²⁺ levels. Results: The amplitude of the calcium current was independent of intracellular Ca²⁺ concentrations. High levels of intracellular Ca²⁺ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca²⁺ by Na⁺ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca²⁺ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ?100 mV for the Ca²⁺ current, but did not reach steady state even at ?150 mV for the Na⁺ current. Conclusions: Ca²⁺ ions facilitate transitions of Ca_v3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor.     

Temperature dependence of T-type calcium channel gating

T-type calcium channel isoforms are expressed in a multitude of tissues and have a key role in a variety of physiological processes. To fully appreciate the physiological role of distinct channel isoforms it is essential to determine their kinetic properties under physiologically relevant conditions. We therefore characterized the gating behavior of expressed rat voltage-dependent calcium channels (Ca(v)) 3.1, Ca(v)3.2, and Ca(v)3.3, as well as human Ca(v)3.3 at 21 degrees C and 37 degrees C in saline that approximates physiological conditions. Exposure to 37 degrees C caused significant increases in the rates of activation, inactivation, and recovery from inactivation, increased the current amplitudes, and induced a hyperpolarizing shift of half-activation for Ca(v)3.1 and Ca(v)3.2. At 37 degrees C the half-inactivation showed a hyperpolarizing shift for Ca(v)3.1 and Ca(v)3.2 and human Ca(v)3.3, but not rat Ca(v)3.3. The observed changes in the kinetics were significant but not identical for the three isoforms, showing that the ability of T-type channels to conduct calcium varies with both channel isoform and temperature.     

Temperature dependence of Cav1.4 calcium channel gating

The CACNA1F gene encodes the pore-forming subunit of the L-type Cav1.4 voltage-gated calcium channel (VGCC) and plays a central role in tonic vesicular release at photoreceptor ribbon synapses. The main objective of this study was to examine the effects of temperature on human Cav1.4 cDNA clone VGCCs. With 20 mM Ba2+ as charge carrier, increasing the temperature from 23 degrees C to 37 degrees C increases whole-cell conductance, shifts the voltage-dependence of activation to more hyperpolarized voltages, and accelerates the degree of recovery from inactivation over a given time, but does not significantly alter the half-inactivation potential (Vh). The window current for Cav1.4 was also shifted to more hyperpolarized voltages, observable from approximately -35 mV to +20 mV at 37 degrees C in 20 mM Ba2+. Several comparable results were observed when characterizing Cav1.2 at temperatures ranging from 23 degrees C to 37 degrees C. However, one difference between Cav1.4 and Cav1.2 was the temperature dependence of voltage-dependent inactivation kinetics. Increasing temperature from 23 degrees C to 37 degrees C accelerates Cav1.4 inactivation kinetics approximately 50-fold, whereas Cav1.2 only accelerates approximately 10-fold over the same temperature range. The time constant of inactivation (tauh) temperature coefficient (Q10) was 18.8 for Cav1.4 over a temperature range of 23 degrees to 33 degrees C (corresponding to an activation energy Ea=221 kJ/mol), compared with Cav1.2 with a Q10 of 3 (Ea=90 kJ/mol) recorded under identical conditions. In addition, Cav1.4 was also tested using 2 mM Ca2+ as a charge carrier and similar changes in current-voltage Boltzmann parameters and gating kinetics were observed. Hence, despite the accelerated inactivation kinetics of Cav1.4 channels observed at near physiological temperatures the window current is preserved and could allow for tonic glutamate release from photoreceptors in the retina during dark adapted conditions.     

Role of Domain IV/S4 outermost arginines in gating of T-type calcium channels

The role of the outermost three charged residues of Domain IV/S4 in controlling gating of Ca_v3.2 was investigated using single substitutions of each arginine with glutamine, cysteine, histidine, and lysine in a Flp-In-293 cell line, in which expression levels could be compared. Channel density, based on gating charge measurements, was ~125,000 channels/cell (10 fC/pF), except for R2Q and R3C, which expressed at lower levels. Channels substituted at Arg-1715 (R1C, R1Q, R1H) demonstrated such modest changes that a role in voltage sensing could not be determined. Arg-1718 (R2) made a contribution to activation voltage sensing, and the channel was sensitive to the geometry of side-chain substitutions at this position. Arg-1721 (R3) substitutions produced complex kinetic changes that together suggested that geometry made a larger contribution than charge. Current decay at positive potentials (OI) exponentially approached a constant value for all mutants except R2K channels, which were biphasically dependent on potential. R2K channels also displayed slowed deactivation with reduced voltage dependence despite near control values for conductance. Voltage-dependent accessibility of R to C mutants, evaluated with intracellularly and extracellularly applied methanthiosulfonate (MTS) reagents, showed that both R2 and R3 were exposed only when cells were depolarized, although it was not necessary for channels to open. Together, the data indicate that Domain IV/S4 is an activation domain and is not involved in inactivation from the open state.     

Targeting of CaV3.2 T-type calcium channels in peripheral sensory neurons for the treatment of painful diabetic neuropathy

Pain-sensing sensory neurons (nociceptors) of the dorsal root ganglion (DRG) can become sensitized (hyperexcitable) in response to pathological conditions such as diabetes, which in turn may lead to the development of painful peripheral diabetic neuropathy (PDN). Because of insufficient knowledge about the mechanisms for this hypersensitization, current treatment for painful PDN has been limited to somewhat nonspecific systemic drugs having significant side effects or potential for abuse. Recent studies have established that the Ca_V3.2 isoform of T-channels makes a previously unrecognized contribution to sensitization of pain responses by enhancing excitability of nociceptors in animal models of type 1 and type 2 PDN. Furthermore, it has been reported that the glycosylation inhibitor neuraminidase can inhibit the native and recombinant Ca_V3.2 T-currents in vitro and completely reverse mechanical and thermal hyperalgesia in diabetic animals with PDN in vivo. Understanding details of posttranslational regulation of nociceptive channel activity via glycosylation may facilitate development of novel therapies for treatment of painful PDN. Pharmacological targeting the specific pathogenic mechanism rather than the channel per se may cause fewer side effects and reduce the potential for drug abuse in patients with diabetes.     

Amazing T-type calcium channels: updating functional properties in health and disease

T-type Ca²⁺ channels have gained, 15 years after cloning, an immense interest as novel players in very unexpected cell functions, and its many relations to diseases have been discovered. This special issue provides a state-of-the-art overview on novel functional properties of T-type Ca²⁺ channels, unexpected cellular functions, and most importantly will also summarizes and review the involvement of this “tiny, transient” type of Ca²⁺ channels in several diseases. It is tried to bridge the gap between molecular biophysical properties of T-type Ca²⁺ channels and diseases providing finally a translational view on this amazing ion channel.     

Models of calcium permeation through T-type channels

Ca²⁺ entry is indispensable part of intracellular Ca²⁺ signaling, which is vital for most of cellular functions. Low voltage-activated (LVA or T-type) calcium channels belong to the family of voltage-gated calcium channels (VGCCs) which provide Ca²⁺ entry in response to membrane depolarization. VGCCs are generally characterized by exceptional Ca²⁺ selectivity combined with high permeation rate, thought to be determined by the presence in their selectivity filter of a versatile Ca²⁺ binding site formed by four glutamate residues (EEEE motif). The subfamily of LVA channels includes three members, Cav3.1, Cav3.2 and Cav3.3. They all possess two aspartates instead of glutamates (i.e., EEDD motif) in their selectivity filter and are the least Ca²⁺-selective of all VGCCs. They also have the lowest conductance, weakly discriminate Ca²⁺, Sr²⁺ and Ba²⁺ and demonstrate channel-specific sensitivity to divalent metal blockers, such as Ni²⁺. The available data suggest that EEDD binding site of LVA channels is more rigid compared to EEEE one, and their selectivity permeation and block are determined by two supplementary low-affinity intrapore Ca²⁺ binding sites located above and below EEDD locus. In addition, LVA channels have extracellular metal binding site that allosterically regulates channel’s gating, permeation and block depending on trace metals concentration.     

Modulation of T-type calcium channels by bioactive lipids

T-type calcium channels (T-channels/Ca_V3) have unique biophysical properties allowing a calcium influx at resting membrane potential of most cells. T-channels are ubiquitously expressed in many tissues and contribute to low-threshold spikes and burst firing in central neurons as well as to pacemaker activities in cardiac cells. They also emerged as potential targets to treat cancer and hypertension. Regulation of these channels appears complex, and several studies have indicated that Ca_V3.1, Ca_V3.2, and Ca_V3.3 currents are directly inhibited by multiple endogenous lipids independently of membrane receptors or intracellular pathways. These bioactive lipids include arachidonic acid and ω3 poly-unsaturated fatty acids; the endocannabinoid anandamide and other N-acylethanolamides; the lipoamino-acids and lipo-neurotransmitters; the P450 epoxygenase metabolite 5,6-epoxyeicosatrienoic acid; as well as similar molecules with 18–22 carbons in the alkyl chain. In this review, we summarize evidence for direct effects of these signaling molecules, the molecular mechanisms underlying the current inhibition, and the involved chemical features. The impact of this modulation in physiology and pathophysiology is discussed with a special emphasis on pain aspects and vasodilation. Overall, these data clearly indicate that T-current inhibition is an important mechanism by which bioactive lipids mediate their physiological functions.     

Regulation of T-type calcium channels by Rho-associated kinase

We investigated the regulation of T-type channels by Rho-associated kinase (ROCK). Activation of ROCK via the endogenous ligand lysophosphatidic acid (LPA) reversibly inhibited the peak current amplitudes of rat Ca(v)3.1 and Ca(v)3.3 channels without affecting the voltage dependence of activation or inactivation, whereas Ca(v)3.2 currents showed depolarizing shifts in these parameters. LPA-induced inhibition of Ca(v)3.1 was dependent on intracellular GTP, and was antagonized by treatment with ROCK and RhoA inhibitors, LPA receptor antagonists or GDPssS. Site-directed mutagenesis of the Ca(v)3.1 alpha1 subunit revealed that the ROCK-mediated effects involve two distinct phosphorylation consensus sites in the domain II-III linker. ROCK activation by LPA reduced native T-type currents in Y79 retinoblastoma and in lateral habenular neurons, and upregulated native Ca(v)3.2 current in dorsal root ganglion neurons. Our data suggest that ROCK is an important regulator of T-type calcium channels, with potentially far-reaching implications for multiple cell functions modulated by LPA.     

Presynaptic HCN1 channels regulate Cav3.2 activity and neurotransmission at select cortical synapses

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they are important for regulating synaptic potential integration and plasticity. We found that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. HCN channels inhibited glutamate synaptic release by suppressing the activity of low-threshold voltage-gated T-type (Ca(V)3.2) Ca2(+) channels. Consistent with this, electron microscopy revealed colocalization of presynaptic HCN1 and Ca(V)3.2 subunit. This represents a previously unknown mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.     

Differential discrimination of fast and slow synaptic waveforms by two low-voltage-activated calcium channels

Electrophysiological analysis of human embryonic kidney 293 cells stably expressing recombinant channels was used to compare how the biophysical properties of the low-voltage-activated Ca(2+) channels encoded by the alpha(1G) (Ca(V)3.1) or alpha(1I) (Ca(V)3.3) subunits shape their responses to excitatory synaptic potentials. In medium containing 2 mM extracellular Ca(2+) standard current-voltage relationships demonstrated both channel types to be clearly low-voltage activated with significant slowly activating current responses being observed at -66 mV. At all test potentials examined, activation of Ca(V)3.3 was substantially slower than that of Ca(V)3.1. To probe how these different T-type channels might respond to excitatory postsynaptic potentials (EPSPs), mock EPSPs with different kinetic profiles were created from the sum of exponentials. These waveforms were then used as command templates in voltage-clamp experiments. Ca(V)3.1-mediated channels responded effectively to both rapidly decaying mock EPSPs and slowly decaying EPSPs. In contrast, Ca(V)3.3-mediated channels were poorly gated by rapidly decaying EPSPs but were effectively activated by the more prolonged synaptic response. When activated with mock EPSPs Ca(V)3.3-mediated currents were more resistant to steady-state depolarisation of the pre-stimulus holding potential. Ca(V)3.3 currents were also more resistant to repetitive application of prolonged EPSPs, which caused substantial inactivation of Ca(V)3.1-mediated currents. The addition of a single mock action potential to the peak of a rapidly decaying EPSP voltage-clamp template greatly enhanced the currents produced by either Ca(V)3.1 or Ca(V)3.3-expressing cells. This facilitatory effect was considerably greater for Ca(V)3.3-mediated channels. From these data we suggest that the slow activation kinetics of Ca(V)3.3-mediated T-type channels enable them to respond selectively to either slow or suprathreshold synaptic potentials.     

Expression of voltage sensitive calcium channel (VSCC) L-type Cav1.2 (alpha1C) and T-type Cav3.2 (alpha1H) subunits during mouse bone development.

Voltage-sensitive calcium channels (VSCCs) are key regulators of osteoblast plasma membrane Ca(2+) permeability and are under control of calcitropic hormones. Subtype specific antibodies were used to probe L-type Ca(v)1.2 (alpha(1C)) and T-type Ca(v)3.2 (alpha(1H)) subunit expression during mouse skeletal development. Commencing from E14.5 and continuing through skeletal maturity, immunoreactivity of Ca(v)1.2 (alpha(1C)) subunits was evident in regions of rapid long bone growth, including the perichondrium, periosteum, chondro-osseous junction and trabecular bones. Ca(v)3.2 (alpha(1H)) subunits appeared simultaneously and followed a similar distribution pattern. Both subunits were observed in osteoblasts and chondrocytes under high magnification. Interestingly, Ca(v)3.2 (alpha(1H)) subunits were present, but Ca(v)1.2 (alpha(1C)) subunits were absent from osteocytes. Western Blot and immunohistochemical assessment of in vitro cell culture models of osteogenesis and chondrogenesis confirmed the in vivo observations. We conclude that both L-type Ca(v)1.2 (alpha(1C)) and T-type Ca(v)3.2 (alpha(1H)) VSCCs are dynamically regulated in bones and cartilages during endochondral bone development.