Mapping of dihydropyridine binding residues in a less sensitive invertebrate L-type calcium channel (LCa v 1)

Invertebrate L-type calcium channel, LCa(v) 1, isolated from the pond snail Lymnaea stagnalis is nearly indistinguishable from mammalian Ca(v) 1.2 (α1C) calcium channel in biophysical characteristics observed in vitro. These L-type channels are likely constrained within a narrow range of biophysical parameters to perform similar functions in the snail and mammalian cardiovascular systems. What distinguishes snail and mammalian L-type channels is a difference in dihydropyridine sensitivity: 100 nM isradipine exhibits a significant block of mammalian Ca(v) 1.2 currents without effect on snail LCa(v)1 currents. The native snail channel serves as a valuable surrogate for validating key residue differences identified from previous experimental and molecular modeling work. As predicted, three residue changes in LCa(v)1 (N_3o18, F_3i10, and I_4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q_3o18, Y_3i10, and M_4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N_3o18_Q mutation in LCa(v) 1 channels lowers DHP sensitivity even further and the triple mutation bearing enhanced isradipine sensitivity, still retains a reduced potency of agonist, (S)-Bay K8644.     

Molecular cloning and functional expression of Ca(v)3.1c, a T-type calcium channel from human brain

Low voltage-activated T-type calcium channels are encoded by a family of at least three genes, with additional diversity created by alternative splicing. This study describes the cloning of the human brain alpha1G, which is a novel isoform, Ca(v)3.1c. Comparison of this sequence to genomic sequences deposited in the GenBank allowed us to identify the intron/exon boundaries of the human CACNA1G gene. A full-length cDNA was constructed, then used to generate a stably-transfected mammalian cell line. The resulting currents were analyzed for their voltage- and time-dependent properties. These properties identify this gene as encoding a T-type Ca(2+) channel.     

A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.     

Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels

T-type Ca(2+) currents have been proposed to be involved in the genesis of spike-and-wave discharges, a sign of absence seizures, but direct evidence in vivo to support this hypothesis has been lacking. To address this question, we generated a null mutation of the alpha(1G) subunit of T-type Ca(2+) channels. The thalamocortical relay neurons of the alpha(1G)-deficient mice lacked the burst mode firing of action potentials, whereas they showed the normal pattern of tonic mode firing. The alpha(1G)-deficient thalamus was specifically resistant to the generation of spike-and-wave discharges in response to GABA(B) receptor activation. Thus, the modulation of the intrinsic firing pattern mediated by alpha(1G) T-type Ca(2+) channels plays a critical role in the genesis of absence seizures in the thalamocortical pathway.     

T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na(+) currents as well as transient Ca(2+) currents abolished by the external application of Ni(2+). Biophysical and pharmacological data indicated that the Ca(2+) current is predominantly mediated by T-type (Ca(v)3.2) channels. The number of cells expressing T-type channels and Ca(v)3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca(2+) currents with Ni(2+) induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Ca(v)3.2) Ca(2+) channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Ca(v)3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca(2+) entry mediated by Ca(v)3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.     

Biophysics and structure-function relationship of T-type Ca2+ channels

T-type channels are distinguished among voltage-gated Ca2+ channels by their low voltage thresholds for activation and inactivation, fast inactivation and small single channel conductance in isotonic Ba2+. Detailed biophysical and pharmacological characterization of native T-type channels indicated that these channels represent a heterogeneous family. Cloning of three family members (CaV3.1-3.3) confirmed these observations and allowed the study of the structure-function relationship of these channels. T-type channels are likely heterotetrameric structures consisting of a single polypeptide of four homologous domains (I-IV), each one containing six transmembrane spans (S1-S6), and cytoplasmic N- and C-termini. Structure-function studies have revealed that fast macroscopic inactivation of CaV3.1 is modulated by specific residues in the proximal C-terminus and in the transmembrane domain IIIS6. The particular gating properties within the T-type channel subfamily are determined by several parts of the protein, whereas differences with respect to high-voltage-activated Ca2+ channels are mostly determined by domains I, II and III. Several gating properties are affected by alternative splicing, C-terminal truncations and mutations associated to idiopathic epilepsy. Intriguingly, the aspartate residues of the EEDD locus of the selectivity filter not only determine the permeation properties and the block by Cd2+ and protons, but also activation and deactivation. Mutagenesis has also revealed that the outermost arginines of the S4 segment of domain IV influence the activation of CaV3.2, though no specific voltage-sensing amino acid has yet been properly identified. The selective modulation of CaV3.2 by G-proteins, CaMKII and PKA is determined by the II-III linker and the high-affinity inhibition of CaV3.2 by Ni2+ relies on a histidine residue in the IS3-S4 linker. Certainly, more structure-function studies are needed for a better understanding of T-type channel physiology and the rational design of treatments against T-type channel-related pathologies.     

Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H

Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents.     

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.     

Modulation of Ca(v)3.2 T-type Ca2+ channels by protein kinase C

Although T-type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Ca(v)3.2 T-type channels reconstituted in Xenopus oocytes. Phorbol-12-myristate-13-acetate (PMA) strongly enhanced the amplitude of Ca(v)3.2 channel currents (approximately 3-fold). The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Ca(v)3.2 channel activity via activation of oocyte PKC.     

Chromosomal Location and Some Structural Features of Human Clathrin Light-Chain Genes (CLTA and CLTB)

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to he encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.     

Effect of mibefradil on sodium and calcium currents

Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (Na(V)1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel alpha(1)3.3b + beta(2), the human intestinal L-type Ca2+ channel subunits alpha(1C) + beta(2), or Na(V)1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations > or = 0.1 microM (IC(50) = 0.29 microM), L-type Ca2+ current at > 1 microM (IC(50) = 2.7 microM), and Na+ current at > or = 0.3 microM (IC(50) = 0.98 microM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.     

A novel Ca(V)1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits

Voltage-dependent L-type Ca(2+) (Ca(V)1.2) channels are the principal Ca(2+) entry pathway in arterial myocytes. Ca(V)1.2 channels regulate multiple vascular functions and are implicated in the pathogenesis of human disease, including hypertension. However, the molecular identity of Ca(V)1.2 channels expressed in myocytes of myogenic arteries that regulate vascular pressure and blood flow is unknown. Here, we cloned Ca(V)1.2 subunits from resistance size cerebral arteries and demonstrate that myocytes contain a novel, cysteine rich N terminus that is derived from exon 1 (termed "exon 1c"), which is located within CACNA1C, the Ca(V)1.2 gene. Quantitative PCR revealed that exon 1c was predominant in arterial myocytes, but rare in cardiac myocytes, where exon 1a prevailed. When co-expressed with alpha(2)delta subunits, Ca(V)1.2 channels containing the novel exon 1c-derived N terminus exhibited: 1) smaller whole cell current density, 2) more negative voltages of half activation (V(1/2,act)) and half-inactivation (V(1/2,inact)), and 3) reduced plasma membrane insertion, when compared with channels containing exon 1b. beta(1b) and beta(2a) subunits caused negative shifts in the V(1/2,act) and V(1/2,inact) of exon 1b-containing Ca(V)1.2alpha(1)/alpha(2)delta currents that were larger than those in exon 1c-containing Ca(V)1.2alpha(1)/alpha(2)delta currents. In contrast, beta(3) similarly shifted V(1/2,act) and V(1/2,inact) of currents generated by exon 1b- and exon 1c-containing channels. beta subunits isoform-dependent differences in current inactivation rates were also detected between N-terminal variants. Data indicate that through novel alternative splicing at exon 1, the Ca(V)1.2 N terminus modifies regulation by auxiliary subunits. The novel exon 1c should generate distinct voltage-dependent Ca(2+) entry in arterial myocytes, resulting in tissue-specific Ca(2+) signaling.     

Alternative splicing modulates inactivation of type 1 voltage-gated sodium channels by toggling an amino acid in the first S3-S4 linker

Voltage-gated sodium channels underlie the upstroke of action potentials and are fundamental to neuronal excitability. Small changes in the behavior of these channels are sufficient to change neuronal firing and trigger seizures. These channels are subject to highly conserved alternative splicing, affecting the short linker between the third transmembrane segment (S3) and the voltage sensor (S4) in their first domain. The biophysical consequences of this alternative splicing are incompletely understood. Here we focus on type 1 sodium channels (Nav1.1) that are implicated in human epilepsy. We show that the functional consequences of alternative splicing are highly sensitive to recording conditions, including the identity of the major intracellular anion and the recording temperature. In particular, the inactivation kinetics of channels containing the alternate exon 5N are more sensitive to intracellular fluoride ions and to changing temperature than channels containing exon 5A. Moreover, Nav1.1 channels containing exon 5N recover from inactivation more rapidly at physiological temperatures. Three amino acids differ between exons 5A and 5N. However, the changes in sensitivity and stability of inactivation were reproduced by a single conserved change from aspartate to asparagine in channels containing exon 5A, which was sufficient to make them behave like channels containing the complete exon 5N sequence. These data suggest that splicing at this site can modify the inactivation of sodium channels and reveal a possible interaction between splicing and anti-epileptic drugs that stabilize sodium channel inactivation.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

Alternative splicing of the rat Ca(v)3.3 T-type calcium channel gene produces variants with distinct functional properties(1)

Molecular diversity in T-type Ca(2+) channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Ca(v)3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Ca(v)3.3 gene produces channels with distinct properties.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.