Pore-forming Alpha 1C Subunit Research Articles

BDNF Levels According to Variations in the CACNA1C Gene: Sex-Based Disparity.

The CACNA1C gene encodes the pore-forming alpha-1c subunit of L-type voltage-gated calcium channels. The calcium influx through these channels regulates the transcription of the brain-derived neurotrophic factor (BDNF). Polymorphisms in this gene have been consistently associated with psychiatric disorders, and alterations in BDNF levels are a possible biological mechanism to explain such associations. Here, we sought to investigate the effect of the CACNA1C rs1006737 and rs4765913 polymorphisms and their haplotypes on serum BDNF concentration. We further aim to investigate the regulatory function of these SNPs and the ones linked to them. The study enrolled 641 young adults (362 women and 279 men) in a cross-sectional population-based survey. Linear regression was used to test the effects of polymorphisms and haplotypes on BDNF levels adjusted for potential confounders. Moreover, regulatory putative functional roles were assessed using in silico approach. BDNF levels were not associated with CACNA1C polymorphisms/haplotype in the total sample. When the sample was stratified by sex, checking the effect of polymorphisms on men and women separately, the A-allele of rs4765913 was associated with lower BDNF levels in women compared with the TT genotype (p = 0.010). The AA (rs1006737-rs4765913) haplotype was associated with BDNF levels in opposite directions regarding sex, with lower levels of BDNF in women (p = 0.040) compared to those without this haplotype, while with higher levels in men (p = 0.027). These findings were supported by the presence of regulatory marks only on the male fetal brain. Our results suggest that the BDNF levels regulation may be a potential mechanism underpinning the association between CACNA1C and psychiatric disorders, with a differential role in women and men.

Negative Transcriptional Regulation of Human Colonic Smooth Muscle Cav1.2 Channels by p50 and p65 Subunits of Nuclear Factor-κB

The expression of Cav1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappaB subunits mediates these effects. Primary cultures of human colonic circular smooth muscle cells and muscle strips were used. The messenger RNA and protein expression of the pore-forming alpha1C subunit of Cav1.2 channels decreased time dependently in response to tumor necrosis factor alpha. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha1C. Three putative kappaB binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha1C gene. Progressive 5' deletions of the promoter and point mutations of the kappaB binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha1C. Transient transfection of human colonic circular muscle strips with antisense oligonucleotides to p50 and p65 decreased expression of the 2 nuclear factor-kappaB units and reversed the suppression of alpha1C, as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha. The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha1C subunit of Cav1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappaB must bind concurrently to the two 5' kappaB motifs on the promoter of alpha1C to produce this effect.

New Short Splice Variants of the Human Cardiac Cavβ2 Subunit: REDEFINING THE MAJOR FUNCTIONAL MOTIFS IMPLEMENTED IN MODULATION OF THE Cav1.2 CHANNEL

Two new short splice variants of the Ca2+ channel beta2 subunit were cloned from human heart poly(A)(+) mRNA. The 410-amino acid beta2f subunit is encoded by exons 1A, 2A, 3, 4, 12, 13, and 14 of the human Cavbeta2 gene and lacks the protein kinase A phosphorylation site, the beta-interaction domain (De Waard, M., Pragnell, M., and Campbell, K. P. (1994) Neuron 13, 495-503), 40% of the beta-SH3 domain, and 73% of the guanylate kinase domain of the putative membrane-associated guanylate kinases module (McGee, A. W., Nunziato, D. A., Maltez, J. M., Prehoda, K. E., Pitt, G. S., and Bredt, D. S. (2004) Neuron 42, 89-99), and helix alpha3 of the alpha1-subunit binding pocket (Van Petegem F., Clark, K. A., Chatelain, F. C., and Minor, D. L., Jr. (2004) Nature 429, 671-675). The beta2g transcript has two potential initiation codons. With the second ATG codon, it generates the 164-amino acid beta2Deltag subunit encoded essentially by the distal part of exon 14, and thus beta2Deltag completely lacks any of the above motifs. Immunoprecipitation analysis confirmed stable association of beta2f and beta2Deltag with the alpha1C subunit. The plasma membrane localization of beta2f and beta2Deltag was substantially increased by co-expression of the alpha1C,77 and alpha2delta subunits. In COS1 cells, beta2f and beta2Deltag increased plasma membrane targeting of the pore-forming alpha1C subunit and differentially facilitated (beta2f > beta2Deltag) the voltage gating of otherwise silent Cav1.2 channels. We conclude that it is unlikely that the beta-interaction domain, membrane-associated guanylate kinases module, and the alpha1-subunit binding pocket helix alpha3 are essential for the interaction of the alpha1C and beta2 subunits and suggest that in addition to the alpha1-subunit binding pocket helices alpha5 and alpha8, a yet unresolved C-terminal beta2 region plays a crucial role.

High blood pressure upregulates arterial L-type Ca2+ channels: is membrane depolarization the signal?

Long-lasting Ca2+ (Ca(L)) channels of the Ca(v)1.2 gene family contribute to the pathogenesis of abnormal arterial tone in hypertension. The physiological stimulus that enhances Ca(L) channel current in the vascular smooth muscle cells (VSMCs) remains unknown. The present study investigated if high blood pressure triggers an upregulation of vascular Ca(L) channel protein. Rat aortae were banded between the origins of the left renal (LR) and right renal (RR) arteries to selectively elevate blood pressure in the proximal RR arteries. After 2 days, the immunoreactivity on Western blots corresponding to the pore-forming alpha1C subunit of the Ca(L) channel was increased 3.25-fold in RR compared with LR arteries. This finding persisted at 28 days and was associated with abnormal Ca2+-dependent tone and higher Ca(L) currents in the VSMCs exposed to high pressure. Based on microelectrode studies indicating that RR arteries were depolarized compared with LR arteries, further studies examined if membrane depolarization, an inherent response of VSMCs to high blood pressure, increased alpha1C expression. Isolated rat renal arteries were cultured for 2 days in low K+ (4 mmol/L) or depolarizing high K+ (30 mmol/L) media. Arteries preconditioned in high K+ showed a 5.47-fold increase in alpha1C expression, enhanced Ca(L) channel current, and elevated Ca2+-dependent tone. These findings provide the first direct evidence that high blood pressure upregulates the Ca(L) channel alpha1C subunit in VSMCs in vivo and suggest that membrane depolarization is a potential signal involved in this interaction that may contribute to the development of abnormal vascular tone.

Key roles of Phe1112 and Ser1115 in the pore-forming IIIS5-S6 linker of L-type Ca2+ channel alpha1C subunit (CaV 1.2) in binding of dihydropyridines and action of Ca2+ channel agonists.

Voltage-dependent L-type Ca2+ channels are modulated by the binding of Ca2+ channel antagonists and agonists to the pore-forming alpha1c subunit (CaV 1.2). We recently identified Ser1115 in IIIS5-S6 linker of alpha1C subunit as a critical determinant of the action of 1,4-dihydropyridine agonists. In this study, we applied alanine-scanning mutational analysis in IIIS5-S6 linker of rat brain alpha1C subunit (rbCII) to illustrate the role of pore-forming IIIS5-S6 linker in the action of Ca2+ channel modulators. Ca2+ channel currents through wild-type (rbCII) or mutated alpha1C subunits, transiently expressed in BHK6 cells with beta1a and alpha2/delta subunits, were analyzed. The replacement of Phe1112 by Ala (F1112A) significantly impaired the sensitivity to Ca2+ channel agonists (S)-(-)-Bay k 8644 and FPL-64176, and modestly to 1,4-dihydropyridine (DHP) antagonists. The low sensitivity of F1112A and S1115A to DHP antagonists was consistent with the reduced binding affinity for [3H](+)PN200-110. The replacement of Phe1112 by Tyr, but not by Ala, restored the long openings produced by FPL-64176, thus indicating the critical role of aromatic ring of Phe1112 in the Ca2+ channel agonist action. Interestingly, double-mutant Ca2+ channel (F1112A/S1115A) failed to discriminate between Ca2+ channel agonist (S)-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl] phenyl)-3-pyridine carboxylic acid methyl ester (Bay k 8644) and antagonist (R)-(+)-Bay k 8644 and was blocked by the two enantiomers in an identical manner. These results indicate that both Phe1112 and Ser1115 in linker IIIS5-S6 are required for the action of Ca2+ channel agonists. A model of the DHP receptor is proposed to visualize possible interactions of Phe1112, Ser1115, and other DHP-sensing residues with a typical DHP ligand nifedipine.

Upregulation of L-type Ca2+ channels in mesenteric and skeletal arteries of SHR.

An increased Ca2+ influx attributed to dihydropyridine-sensitive L-type Ca2+ channels has been demonstrated in mesenteric vascular smooth muscle cells of spontaneously hypertensive rats (SHR). This study examined whether an upregulation of the pore-forming alpha1C subunit of the L-type Ca2+ channel underlies this ionic defect. With the use of mesenteric arcade arteries from 12- to 16-week-old SHR and normotensive Wistar Kyoto (WKY) rats, reverse transcriptase-polymerase chain reaction demonstrated an increased level of amplified cDNA corresponding to the alpha1C subunit mRNA in the SHR arteries. Western blots confirmed that the increased mRNA expression was associated with a 3.4-fold increase in the immunoreactive signal of the alpha1C subunit protein in SHR compared with WKY mesenteric arteries, and immunocytochemistry confirmed this abnormality at the single-cell level. Finally, isolated mesenteric arteries from SHR were highly reactive to Bay K8644 and developed anomalous Ca2+-dependent tone, suggesting a functional role for alpha1C subunit upregulation in vascular hyperreactivity. To determine if these Ca2+ channel abnormalities extended to the SHR skeletal muscle bed, we repeated a similar series of studies in WKY and SHR hind limb arteries. Skeletal muscle arteries from SHR also expressed higher levels of alpha1C subunit mRNA and protein than WKY arteries and developed anomalous Ca2+-dependent tone attributed to L-type Ca2+ channels. Our data provide the first evidence that the alpha1C subunit mRNA and protein are upregulated in SHR arteries and that the increased numbers of L-type Ca2+ channel pores are associated with the generation of abnormal vascular tone.

New Molecular Determinant for Inactivation of the Human L-Type α 1C Ca 2+ Channel

Molecular cloning of the human fibroblast Ca2+ channel pore-forming alpha1C subunit revealed (Soldatov, 1992. Proc. Natl. Acad. Sci. USA 89:4628-4632) a naturally occurring mutation g2254 --> a that causes the replacement of the conservative alanine for threonine at the position 752 at the cytoplasmic end of transmembrane segment IIS6. Using stably transfected HEK293 cell lines, we have compared electrophysiological properties of the conventional alpha(1C,77) human recombinant L-type Ca2+ channel with those of its mutated isoform alpha(1C,94) containing the A752T replacement. Comparative quantification of steady-state availability of the current carried by alpha(1C,94) and alpha(1C,77) showed that A752T mutation prevented a large (approximately 25%) fraction of the current carried by Ca2+ or Ba2+ from fully inactivating. This mutation, however, did not appear to alter significantly the Ca2+-dependence and kinetics of decay of the inactivating fraction of the current or its voltage-dependence. The data suggests that Ala752 at the cytoplasmic end of IIS6 might serve as a molecular determinant of the Ca2+ channel inactivation, possibly regulating the voltage-dependence of its availability.

Complexes of the α1C and β Subunits Generate the Necessary Signal for Membrane Targeting of Class C L-type Calcium Channels

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha1C subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha1C subunit with either the wild-type beta2a subunit, a palmitoylation-deficient beta2a(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta1b, beta3, and beta4 subunits) resulted in the redistribution of both the alpha1C and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha1C was co-expressed with an N- and C-terminal truncated mutant beta2a containing only the central conserved regions. However, when the alpha1C subunit was co-expressed with an alpha1 beta interaction-deficient mutant, beta2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta2a that was unable to interact with the alpha1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha1C subunit with the largely peripheral accessory alpha2 delta subunit was ineffective in recruiting alpha1C to the plasma membrane, while co-distribution of all three subunits was observed when beta2a was co-expressed with the alpha1C and alpha2 delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha1 and beta subunits.

Promotion and Inhibition of L-type Ca2+Channel Facilitation by Distinct Domains of the β Subunit

Ca2+ current potentiation by conditioning depolarization is a general mechanism by which excitable cells can control the level of Ca2+ entry during repetitive depolarizations. Several types of Ca2+ channels are sensitive to conditioning depolarization, however, using clearly distinguishable mechanisms. In the case of L-type Ca2+ channels, prepulse-induced current facilitation can only be recorded when the pore-forming alpha1C subunit is coexpressed with the auxiliary beta1, beta3, or beta4, but not beta2, subunit. These four beta subunits are composed of two conserved domains surrounded by central, N-terminal, and C-terminal variable regions. Using different deleted and chimeric forms of the beta1 and beta2 subunits, we have mapped essential sequences for L-type Ca2+ channel facilitation. A first sequence, located in the second conserved domain of all beta subunits, is responsible for the promotion of current facilitation by the beta subunit. A second sequence of 16 amino acids, located on the N-terminal tail of the beta2 subunit, induces a transferable block of L-type current facilitation. Site-specific mutations reveal the essential inhibitory role played by three positive charges on this segment. The lack of prepulse-induced current facilitation recorded with some truncated forms of the beta2 subunit suggests the existence of an additional inhibitory sequence in the beta2 subunit.

Molecular Structures Involved in L-type Calcium Channel Inactivation: ROLE OF THE CARBOXYL-TERMINAL REGION ENCODED BY EXONS 40-42 IN α1C SUBUNIT IN THE KINETICS AND Ca2+ DEPENDENCE OF INACTIVATION

The pore-forming alpha1C subunit is the principal component of the voltage-sensitive L-type Ca2+ channel. It has a long cytoplasmic carboxyl-terminal tail playing a critical role in channel gating. The expression of alpha1C subunits is characterized by alternative splicing, which generates its multiple isoforms. cDNA cloning points to a diversity of human hippocampus alpha1C transcripts in the region of exons 40-43 that encode a part of the 662-amino acid carboxyl terminus. We compared electrophysiological properties of the well defined 2138-amino acid alpha1C,77 channel isoform with two splice variants, alpha1C,72 and alpha1C,86. They contain alterations in the carboxyl terminus due to alternative splicing of exons 40-42. The 2157-amino acid alpha1C,72 isoform contains an insertion of 19 amino acids at position 1575. The 2139-amino acid alpha1C,86 has 80 amino acids replaced in positions 1572-1651 of alpha1C,77 by a non-identical sequence of 81 amino acids. When expressed in Xenopus oocytes, all three splice variants retained high sensitivity toward dihydropyridine blockers but showed large differences in gating properties. Unlike alpha1C,77 and alpha1C,72, Ba2+ currents (IBa) through alpha1C,86 inactivated 8-10 times faster at +20 mV, and its inactivation rate was strongly voltage-dependent. Compared to alpha1C,77, the inactivation curves of IBa through alpha1C,86 and alpha1C,72 channels were shifted toward more negative voltages by 11 and 6 mV, respectively. Unlike alpha1C,77 and alpha1C,72, the alpha1C,86 channel lacks a Ca2+-dependent component of inactivation. Thus the segment 1572-1651 of the cytoplasmic tail of alpha1C is critical for the kinetics as well as for the Ca2+ and voltage dependence of L-type Ca2+ channel gating.