Splice-variant changes of the CaV3.2 T-type calcium channel mediate voltage-dependent facilitation and associate with cardiac hypertrophy and development

Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (Ca_V3.1, Ca_V3.2, Ca_V3.3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize Ca_V3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length Ca_V3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac Ca_V3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression of Ca_V3.2(−25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (−25) exon variants, (3) in the adult stage of hypertensive rats there is both an increase in overall Ca_V3.2 expression and a shift towards expression of Ca_V3.2(+25) containing channels as the predominant form and (4) alternative splicing confers a variant-specific voltage-dependent facilitation of Ca_V3.2 channels. We conclude that Ca_V3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.Key words: voltage-dependent facilitation, alternative splicing, T-type calcium channel, hypertension, cardiac hypertrophy     

Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria

Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca²⁺ channels (Ca_V3.1, Ca_V3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Ca_v3.1 and Ca_V3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca²⁺ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both Ca_V3.1 and Ca_V3.2 in co-immunoprecipitation experiments. T-type Ca²⁺ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify Ca_V3.1 and Ca_V3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca²⁺ is essential for cardiomyocyte function, and alterations in proteins that regulate Ca²⁺ influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca²⁺ channels are one pathway of Ca²⁺ entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Ca_v3.1 (α1G) and Ca_v3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca²⁺ channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Ca_v3.1 and Ca_v3.2 Ca²⁺ channel expression in the pathological setting. The putative association of T-type Ca²⁺ channels with Ca²⁺ dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

A novel disease connection for TRPM2 channels

Low voltage-activated (LVA) T-type calcium channels play critical roles in the excitability of many cell types and are a focus of research aimed both at understanding the physiological basis of calcium channel-dependent signaling and the underlying pathophysiology associated with hyperexcitability disorders such as epilepsy. These channels play a critical role towards neuronal firing in both conducting calcium ions during action potentials and also in switching neurons between distinct modes of firing. In this review the properties of the Ca_V3.1, Ca_V3.2 and Ca_V3.3 T-type channel isoforms is discussed in relation to their individual contributions to action potentials during burst and tonic firing states as well their roles in switching between firing states.     

T-type Channels Become Highly Permeable to Sodium Ions Using an Alternative Extracellular Turret Region (S5-P) Outside the Selectivity Filter

T-type (Ca_v3) channels are categorized as calcium channels, but invertebrate ones can be highly sodium-selective channels. We illustrate that the snail LCa_v3 T-type channel becomes highly sodium-permeable through exon splicing of an extracellular turret and descending helix in domain II of the four-domain Ca_v3 channel. Highly sodium-permeable T-type channels are generated without altering the invariant ring of charged residues in the selectivity filter that governs calcium selectivity in calcium channels. The highly sodium-permeant T-type channel expresses in the brain and is the only splice isoform expressed in the snail heart. This unique splicing of turret residues offers T-type channels a capacity to serve as a pacemaking sodium current in the primitive heart and brain in lieu of Na_v1-type sodium channels and to substitute for voltage-gated sodium channels lacking in many invertebrates. T-type channels would also contribute substantially to sodium leak conductances at rest in invertebrates because of their large window currents.     

CaV1.2/CaV3.x channels mediate divergent vasomotor responses in human cerebral arteries

The regulation of arterial tone is critical in the spatial and temporal control of cerebral blood flow. Voltage-gated Ca²⁺ (Ca_V) channels are key regulators of excitation–contraction coupling in arterial smooth muscle, and thereby of arterial tone. Although L- and T-type Ca_V channels have been identified in rodent smooth muscle, little is known about the expression and function of specific Ca_V subtypes in human arteries. Here, we determined which Ca_V subtypes are present in human cerebral arteries and defined their roles in determining arterial tone. Quantitative polymerase chain reaction and Western blot analysis, respectively, identified mRNA and protein for L- and T-type channels in smooth muscle of cerebral arteries harvested from patients undergoing resection surgery. Analogous to rodents, Ca_V1.2 (L-type) and Ca_V3.2 (T-type) α₁ subunits were expressed in human cerebral arterial smooth muscle; intriguingly, the Ca_V3.1 (T-type) subtype present in rodents was replaced with a different T-type isoform, Ca_V3.3, in humans. Using established pharmacological and electrophysiological tools, we separated and characterized the unique profiles of Ca²⁺ channel subtypes. Pressurized vessel myography identified a key role for Ca_V1.2 and Ca_V3.3 channels in mediating cerebral arterial constriction, with the former and latter predominating at higher and lower intraluminal pressures, respectively. In contrast, Ca_V3.2 antagonized arterial tone through downstream regulation of the large-conductance Ca²⁺-activated K⁺ channel. Computational analysis indicated that each Ca²⁺ channel subtype will uniquely contribute to the dynamic regulation of cerebral blood flow. In conclusion, this study documents the expression of three distinct Ca²⁺ channel subtypes in human cerebral arteries and further shows how they act together to orchestrate arterial tone.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca (2+) is essential for cardiomyocyte function, and alterations in proteins that regulate Ca (2+) influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca (2+) channels are one pathway of Ca (2+) entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Cav3.1 (α1G) and Cav3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca (2+) channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Cav3.1 and Cav3.2 Ca (2+) channel expression in the pathological setting. The putative association of T-type Ca (2+) channels with Ca (2+) dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure

Recently, we reported that homozygous deletion of alternative exon 33 of Ca_V1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased Ca_V1.2_Δ33 I_CaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33^+/? cardiomyocytes showed similar Ca_V1.2 channel properties as wild-type cardiomyocyte, even though Ca_V1.2_Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of Ca_V1.2 exon 33. (149 words)     

Labelling of the 3D structure of the cardiac L-type voltage-gated calcium channel

Voltage-gated calcium channels (VGCCs) regulate calcium influx into all excitable cells. In the heart, the main calcium channels are the L-type VGCCs (LTCCs). These are localised to the sarcolemmal membrane, and are hetero-oligomeric complexes comprised of three non-covalently associated polypeptides; alpha1 (Ca_V1.2), alpha2delta and beta. We recently reported the 3D structure for a monomeric form of the cardiac LTCC1 using electron microscopy and single particle analysis. We also determined the first medium/low resolution structure of a T-type voltage gated calcium channel (Ca_V3.1) polypeptide. We identified the transmembrane and cytoplasmic domains of the T-type channel using labelling studies to determine the position of the C-terminus. By modelling of the Ca_V3.1 structure (comparable at these resolutions to Ca_V1.2) into the cardiac LTCC volume, we were able to delineate the subunit boundaries of the cardiac LTCC, leading to a proposal for a putative orientation of the LTCC with respect to the membrane bilayer. We have now extended these studies to include labelling of the extracellular alpha2 polypeptide using affinity purified antibodies raised against the Von Willebrand Factor A (VWA) domain and calmodulin-gold labelling of the C-terminus of Ca_V1.2. These data provide further support for the proposed orientation of the 3D structure of the cardiac LTCC.     

Structural and biophysical determinants of single CaV3.1 and CaV3.2 T-type calcium channel inhibition by N2O

We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels Ca_V3.1 and Ca_V3.2 by nitrous oxide (N₂O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N₂O significantly inhibited Ca_V3.2, and – less pronounced – Ca_V3.1. A Ca_V3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N₂O reduced average peak current of Ca_V3.2 by decreasing open probability and open time duration. Effects on Ca_V3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single Ca_V3.1 channels were significantly less active than Ca_V3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within Ca_V3.2 (H191), are responsible for the subtype-prevalent N₂O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N₂O on Ca_V3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant Ca_V3.1.     

Splice-variant-specific effects of primary aldosteronism point mutations on human CaV3.2 calcium channels

Ca_V3.2 calcium channels play important roles in both neural excitability and aldosterone secretion. Recent clinical studies found four germline mutations (S196 L, M1549I, V1951E and P2083 L) in Ca_V3.2 channels. All four mutations caused primary aldosteronism (PA), while only the M1549I mutation resulted in obvious neural malfunctions besides PA. In human, there are two major Ca_V3.2 channel gene (CACNA1H) splice variants, either with or without exon 26. In this study, we tested the expression of the two CACNA1H splice variants in zona glomerulosa (ZG) cells of human adrenal cortex and the possibility that Ca_V3.2 (-26) and Ca_V3.2 (+26) channels have different functional responses to the four PA mutations. We found that human ZG cells only express long form Ca_V3.2(+26) channels. The M1549I mutation slowed the inactivation of Ca_V3.2(+26) more than 5 fold, and Ca_V3.2(-26) more than 2 fold. The S196 L, V1951E and P2083 L mutations accelerated channel recovery from inactivation for Ca_V3.2(+26), but not Ca_V3.2(-26) channels. All four mutations resulted in gain of function of Ca_V3.2(+26) channels, leading to overproduction of aldosterone. In conclusion, the four PA mutations caused more profound changes on Ca_V3.2 (+26) currents than on Ca_V3.2 (-26) currents, and except the M1549I mutation, the S196 L, V1951E and P2083 L have little effect on the electrophysiological properties of Ca_V3.2(-26) currents, which may partially explain the limitation of the phenotype associated with the V1951E, S196 L and P2083 L germline mutations to PA.     

T-type Ca2+ channels and autoregulation of local blood flow

L-type voltage gated Ca²⁺ channels are considered to be the primary source of calcium influx during the myogenic response. However, many vascular beds also express T-type voltage gated Ca²⁺ channels. Recent studies suggest that these channels may also play a role in autoregulation. At low pressures (40–80 mmHg) T-type channels affect myogenic responses in cerebral and mesenteric vascular beds. T-type channels also seem to be involved in skeletal muscle autoregulation. This review discusses the expression and role of T-type voltage gated Ca²⁺ channels in the autoregulation of several different vascular beds. Lack of specific pharmacological inhibitors has been a huge challenge in the field. Now the research has been strengthened by genetically modified models such as mice lacking expression of T-type voltage gated Ca²⁺ channels (Ca_V3.1 and Ca_V3.2). Hopefully, these new tools will help further elucidate the role of voltage gated T-type Ca²⁺ channels in autoregulation and vascular function.     

Synthesis and evaluation of aminobenzothiazoles as blockers of N- and T-type calcium channels

Both N- and T-type calcium ion channels have been implicated in pain transmission and the N-type channel is a well-validated target for the treatment of neuropathic pain. An SAR investigation of a series of substituted aminobenzothiazoles identified a subset of five compounds with comparable activity to the positive control Z160 in a FLIPR-based intracellular calcium response assay measuring potency at both Ca_V2.2 and Ca_V3.2 channels. These compounds may form the basis for the development of drug leads and tool compounds for assessing in vivo effects of variable modulation of Ca_V2.2 and Ca_V3.2 channels.     

Role of putative voltage-sensor countercharge D4 in regulating gating properties of CaV1.2 and CaV1.3 calcium channels

Voltage-dependent calcium channels (Ca_V) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of Ca_V1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of Ca_V1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of Ca_V1.3 and in VSDs II and IV of Ca_V1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of Ca_V1.3 and in two splice variants of Ca_V1.2. In both channels the D4N (VSD IV) mutation resulted in a ?5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in Ca_V1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined Ca_V1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle Ca_V1.1 channel.     

GABAB receptors suppress burst-firing in reticular thalamic neurons

Burst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN. The role of this putative negative feedback upon the RTN itself is less well understood, although is hypothesized to induce de-synchronization of RTN neuron firing leading to the suppression of TC oscillations. Here we tested two hypotheses concerning possible mechanisms underlying TC oscillation modulation. Firstly, we assessed the burst-firing behavior of RTN neurons in response to GABA_B receptor activation using acute brain slices. The selective GABA_B receptor agonist baclofen was found to induce suppression of burst-firing concurrent with effects on membrane input resistance. Secondly, RTN neurons express Ca_V3.2 and Ca_V3.3 T-type calcium channel isoforms known to contribute toward TC burst-firing and we examined the modulation of these channels by GABA_B receptor activation. Utilizing exogenously expressed T-type channels we assessed whether GABA_B receptor activation could directly alter T-type calcium channel properties. Overall, GABA_B receptor activation had only modest effects on Ca_V3.2 and Ca_V3.3 isoforms. The only effect that could be predicted to suppress burst-firing was a hyperpolarized shift in the voltage-dependence of inactivation, potentially causing lower channel availability at membrane potentials critical for burst-firing. Conversely, other effects observed such as a hyperpolarized shift in the voltage-dependence of activation of both Ca_V3.2 and Ca_V3.3 as well as increased time constant of activation of the Ca_V3.3 isoform would be expected to enhance burst-firing. Together, we hypothesize that GABA_B receptor activation mediates multiple downstream effectors that combined act to suppress burst-firing within the RTN. It appears unlikely that direct GABA_B receptor-mediated modulation of T-type calcium channels is the major mechanistic contributor to this suppression.     

Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation

Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Ca_v3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCa_v3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCa_v3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.     

Functional Expression of T-Type Ca2+ Channels in Spinal Motoneurons of the Adult Turtle

Voltage-gated Ca²⁺ (Ca_V) channels are transmembrane proteins comprising three subfamilies named Ca_V1, Ca_V2 and Ca_V3. The Ca_V3 channel subfamily groups the low-voltage activated Ca²⁺ channels (LVA or T-type) a significant role in regulating neuronal excitability. Ca_V3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR). In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, Ca_V3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of Ca_V3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni²⁺ and NNC55-0396, two antagonists of Ca_V3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the Ca_V3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of Ca_V3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.     

Redox Mechanism of S-Nitrosothiol Modulation of Neuronal CaV3.2 T-Type Calcium Channels

T-type calcium channels in the dorsal root ganglia (DRG) have a central function in tuning neuronal excitability and are implicated in sensory processing including pain. Previous studies have implicated redox agents in control of T-channel activity; however, the mechanisms involved are not completely understood. Here, we recorded T-type calcium currents from acutely dissociated DRG neurons from young rats and investigated the mechanisms of Ca_V3.2 T-type channel modulation by S-nitrosothiols (SNOs). We found that extracellular application of S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-penicillamine rapidly reduced T-type current amplitudes. GSNO did not affect voltage dependence of steady-state inactivation and macroscopic current kinetics of T-type channels. The effects of GSNO were abolished by pretreatment of the cells with N-ethylmaleimide, an irreversible alkylating agent, but not by pretreatment with 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one, a specific soluble guanylyl cyclase inhibitor, suggesting a potential effect of GSNO on putative extracellular thiol residues on T-type channels. Expression of wild-type Ca_V3.2 channels or a quadruple Cys-Ala mutant in human embryonic kidney cells revealed that Cys residues in repeats I and II on the extracellular face of the channel were required for channel inhibition by GSNO. We propose that SNO-related molecules in vivo may lead to alterations of T-type channel-dependent neuronal excitability in sensory neurons and in the central nervous system in both physiological and pathological conditions such as neuronal ischemia/hypoxia.