1C Subunit Research Articles

Bilateral molecular defects in infertile men with left varicocele

Objective: Varicocele are a common cause of male infertility but the underlying mechanism remains unclear. Varicocele affect maximally the latest stages of spermatogenesis. Bilateral degenerative testicular changes are frequently observed in infertile men with left varicocele (LV), suggesting that factors in addition to varicocele contribute to the production of the infertile state. One co-factor is an elevation in left testicular cadmium (Cd2+) levels, leading sequentially to aberrant splicing (deletions of exons 7 and/or 8) of testis-specific L-type calcium channel (L-VDCC) 1 subunit mRNA (controlling metal ion sensitivity), loss of actin and increased germ cell apoptosis. Since calcium is involved in these three events, an alteration in calcium homeostasis is implicated in impaired spermatogenesis in LV. Our goal was to determine whether affects of altered calcium homeostasis appear in the contralateral testis. Design: Cd2+ levels, L-VDCC 1C mRNA sequence, the presence of cAMP response element modulator (CREM) and transition protein-1 (TP1) mRNAs, CREM antigenic epitopes and apoptosis were assessed in bilateral testicular biopsies (TB) from men with LV and men with bilateral varicocele (BLV). TB from men with obstructive azoospermia (OA) served as control. Materials/Methods: Under an IRB-approved protocol, TB were obtained by percutaneous needle aspiration biopsy (BLV, n = 16; LV, n = 17; OA, n = 6). Cd2+ levels were determined by atomic absorption. RNA from each biopsy was used as a template in reverse transcription-polymerase chain reactions (RT-PCR) with primers designed to amplify (1) L-VDCC 1C subunit exons 6–9, (2) transactivator form of the transcription factor CREM, CREM, and (3) exons 1–2 of TP1 (a marker for completion of meiosis and the presence of round spermatids). Apoptosis in TB sections was quantified using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL). CREM antigenic epitopes were localized in serial sections by immunohistochemistry. Results: Cd2+ concentrations in left and right TB from individual patients were similar (Signed rank test, P = 0.760, NS). Both LV and BLV TB could be divided into two subgroups (“normal” and “high”) using an OA TB threshold value (“normal”, <0.368 ng/mg dry wt). Comparing the LV groups with the corresponding BLV groups, Cd2+ concentrations did not differ (Mann-Whitney test, P = 0.692, NS). In all cases, elevated Cd2+ levels correlated with increased germ cell apoptosis (t-test, p <0.0001) and with deletions in exons 7 and/or 8 L-VDCC 1C transcripts (t-tests; left testis, p <0.002; right testis, p <0.0008). CREM protein and transcripts were not detectable in left or right testes in LV or BLV from subjects with “high” Cd2+ levels but, unexpectedly, TP1 transcripts were present. Conclusions: These data indicate the presence of bilateral molecular defects in men with LV. The histological findings in these TB are reminiscent of those in CREM knock-out mice. CREM is a key transcriptional regulator of post-meiotic gene expression which is activated by calcium-modulated phosphorylation. By analogy to CREM knock-outs, the absence of CREM suggests that modulation of calcium homeostasis may be responsible for reduction in sperm number, motility and morphology seen in varicocele as well as arrest of spermatogenesis primarily at the spermatid stage. Supported by: NIH Grant No. ES 10496 to SB.

A new promoter for α 1C subunit of human L-type cardiac calcium channel Ca V1.2

The cardiac Ca channel known as α 1C or Ca V1.2 is shown to express a new longer first exon equivalent to that formerly reported in rabbit heart or rat aorta. Ribonuclease protection assay indicates that this exon is found in the majority of Ca V1.2 transcripts in human heart RNA. The presence of this exon also suggests that expression of this transcript is driven by a promoter immediately upstream of this exon and its 5 ′ untranslated region. The putative promoter exhibits 69% homology to its rat counterpart and displays functional promoter activity when transfected into heart cells in culture in luciferase-expressing constructs.

A 27 bp cis-acting sequence is essential for L-type calcium channel α 1C subunit expression in vascular smooth muscle cells

Expression of L-type calcium channels in cardiac myocytes and vascular smooth muscle cells (VSMC) critically regulates the contractile state of these cells. In order to discover the elements in the promoter region of the Ca v1.2 gene encoding the vascular/cardiac calcium channel α 1C subunit that are important for the basal gene expression, ∼2 kb of the 5′-flanking sequence of the Ca v1.2 gene has been cloned in our lab. In this study, using various lengths of the 5′-flanking DNA fused with a luciferase gene as a reporter, we have defined a 493-bp fragment of the cis-regulatory DNA which carries the majority of promoter activity in pulmonary artery smooth muscle (PAC1) cells. DNase I footprinting analysis of this 493-bp DNA using nuclear extracts from PAC1 cells revealed a 27-bp DNA sequence that contains a c-Ets like motif (CAGGATGC). Mutation of the Ets-like site and the respective flanking sequence within the DNase I footprinting protection region induced a marked change in the promoter activity in PAC1 cells. Electrophoretic mobility shift assays (EMSA) confirmed the presence of specific binding factor(s) in PAC1 cells' nuclear extracts for this 27-bp DNA. Competition studies with the wild-type and mutated DNA fragments established the importance of the 27 bp DNA sequence for high-affinity binding of the nuclear proteins to the promoter. We conclude that there is a 27 bp region in the promoter of the Ca v1.2 gene to which nuclear proteins from VSMC bind and strongly regulate the basal promoter activity.

A mutation in the β interaction domain of the Ca 2+ channel α 1C subunit reduces the affinity of the (+)-[ 3H]isradipine binding site

The molecular mechanisms of how α 1 and β subunits of voltage-gated Ca 2+ channels interact with one another are still controversial. Here we show that despite a mutation in the β interaction domain that has previously been shown to disrupt binding, α 1CY467S and β 1a-myc still formed immunoprecipitable complexes when coexpressed in tsA201 cells. However, the α 1CY467S–β 1a-myc complexes had a decreased affinity to (+)-[ 3H]isradipine. This indicates that the β interaction domain in the I–II loop of the α 1 subunit is not merely an anchor required for the functional interaction of the two Ca 2+ channel subunits but is itself part of the effector pathway for β-induced channel modulation.

Changes in cardiac myocyte morphology alter the properties of voltage-gated ion channels.

The goal of this study was to determine if the properties of the transient outward potassium (I(to)), TTX-resistant sodium (I(Na)) and L-type calcium (I(Ca)) currents are altered during changes in cardiac cell shape. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on either non-aligned or aligned collagen thin gels. In contrast to the flat, stellar-shaped myocytes obtained when the cells are plated on non-aligned collagen gels, myocytes plated on aligned gels display an elongated, rod-like shape. Ion channel expression was measured using the whole-cell arrangement of the patch clamp technique and Western blot analysis. Peak values for I(to), I(Na) and I(Ca) were 9+/-1, 71+/-13 and 7+/-1 pA/pF, respectively, in the flat cells, and increased to 21+/-2, 190+/-26 and 13+/-1 pA/pF, respectively, in the aligned cells. Application of forskolin (2 microM) and 3-isobutyl-1-methylxanthine (100 microM) resulted in a 101+/-18% increase in I(Ca) in the flat cells, but increased the current by only 43+/-9% in the aligned cells. Internal dialysis of the myocytes with cAMP strongly increased the peak I(Ca) in the flat cells, but caused no significant change in the aligned cells. While both basal and forskolin-stimulated levels of cAMP were the same in the two cell morphologies, the expression of the calcium channel alpha(1C) subunit was increased in the aligned cells. The expression and regulatory properties of voltage-gated calcium channels are modified during changes in neonatal rat myocyte shape.

Altered expression of voltage-dependent calcium channel α 1 subunits in temporal lobe epilepsy with Ammon’s horn sclerosis

Voltage-dependent calcium channels, the initial components in the calcium signalling cascade, are increasingly being recognised as relevant factors in the pathology of epilepsy. To further characterise their role in temporal lobe epilepsy associated with Ammon’s horn sclerosis, we investigated the immunohistochemical distribution of five different voltage-dependent calcium channel α 1 subunits (α 1A, α 1B, α 1C, α 1D, α 1E) in 14 hippocampal specimens of patients with Ammon’s horn sclerosis in comparison with eight autopsy control cases. In epilepsy specimens an increased immunoreactivity was observed for α 1A, α 1B, α 1D and α 1E in the neuropil of the dentate gyrus molecular layer. Dentate gyrus granule cells and residual CA3 pyramidal neurones showed enhanced immunoreactivity for α 1A, while labelling of these neurones was decreased for α 1C. Astrocytes in Ammon’s horn sclerosis specimens were strongly immunoreactive for the α 1C subunit contrasting with an absent astrocytic α 1C labelling in controls. Our results suggest that the expression of calcium channels in neurones and glial cells is dynamically regulated in temporal lobe epilepsy, supporting the relevance of calcium signalling pathways for this disease.

Molecular Determinants of Voltage-dependent Slow Inactivation of the Ca2+ Channel

Ba(2+) current through the L-type Ca(2+) channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the alpha(1C) subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba(2+) current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed approximately 45% of the sustained Ba(2+) or Ca(2+) current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (pi(f) approximately 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca(2+)-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca(2+) sensors of inactivation.

Up-regulation of L-type Voltage-dependent Calcium Channels after Long Term Exposure to Nicotine in Cerebral Cortical Neurons

Effects of long term (72-h) exposure to low concentration (0.1 mum) of nicotine on various types of voltage-dependent Ca(2+) channels (VDCCs) and neuronal nicotinic acetylcholine receptors (nnAChRs) were examined using primary cultures of mouse cerebral cortical neurons. High potassium (30 mm KCl)-stimulated (45)Ca(2+) influx into the neurons increased with increasing the duration of nicotine exposure and its concentrations. The maximal increase of the KCl-stimulated (45)Ca(2+) influx was found 24 h after the initiation of exposure and thereafter maintained up to 72 h. This enhancement of KCl-induced (45)Ca(2+) influx after 72-h exposure to 0.1 mum nicotine was completely abolished by concomitant exposure with mecamylamine, an inhibitor for nnAChRs. Only the component of the KCl-induced (45)Ca(2+) influx observed after long term exposure to nicotine, which was sensitive to nifedipine, an inhibitor of L-type VDCCs, was facilitated, while the (45)Ca(2+) influx through P/Q- and N-type VDCCs showed no changes. Moreover, enhanced immunoreactivity against antibody for the alpha(1C) subunit of L-type VDCCs was recognized, whereas no changes in immunoreactivities against antibodies for alpha(1A) and alpha(1B) subunits of other types of VDCCs were noted. In addition, a Western blot analysis showed an increase of immunoreactivities against antibodies for alpha(1D) and alpha(2)/delta(1), and expression of mRNA for L-type VDCC subunit, alpha(1F), was also enhanced, although beta(4) mRNA expression was not changed. Whole cell patch clamp analysis revealed that the increase of the amplitude of Ba(2+) currents was also recognized in the neurons exposed to nicotine, and nicardipine reduced this increased amplitude to the level of the amplitude detected in nontreated neurons with nicardipine. The up-regulation of alpha(4) and beta(2) subunits, but not the alpha(3) subunit of nnAChRs, was also noted after the nicotine exposure when examining by the Western blot analysis. Taken together, these results indicate that the long term exposure of the neurons to a low concentration of nicotine induces both increased (45)Ca(2+) influx through up-regulated L-type VDCCs and nnAChR up-regulation.

Compensatory electrical remodeling in hearts of transgenic mice that overexpress the Ca2+ channel alpha 1C subunit

Compensatory electrical remodeling in hearts of transgenic mice that overexpress the Ca2+ channel alpha 1C subunit

Mechanism of down-regulation of L-type Ca(2+) channel in the proliferating smooth muscle cells of rat aorta.

The mechanism of down-regulation of L-type Ca(2+) channel (L-VOC) was investigated in rat aortic smooth muscle cells in primary culture. On culture days 3-5, the cells actively incorporated the 5-bromo-2'-deoxy-uridine (BrdU), and did not respond to K(+) depolarization nor express alpha(1C) subunit of L-VOC. At confluence on day 8, BrdU incorporation decreased, and the cells up-regulated alpha(1C) subunit mRNA, expressed alpha(1C) subunit protein at cell periphery, and responded to K(+) depolarization. Treating the proliferating cells on day 3 with serum-free media or 10 microM PD98059, a MAP kinase kinase inhibitor, for 2 days induced the expression of alpha(1C) subunit protein and the responsiveness to K(+) depolarization. However, the serum starvation, but not PD98059, decreased the BrdU incorporation and increased the alpha(1C) subunit mRNA. It is concluded that the expression of L-VOC is substantially suppressed in the proliferating cells due to two mechanisms; a MAP kinase-mediated post-transcriptional down-regulation and the transcriptional down-regulation by additional mitogenic signals.

The involvement of L-type Ca(2+) channels in the relaxant effects of the ATP-sensitive K(+) channel opener ZD6169 on pig urethral smooth muscle.

1. The effects of ZD6169, a novel ATP-sensitive K(+) channel (K(ATP) channel) opener, were investigated on membrane currents in isolated myocytes using patch-clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle. 2. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (< or =3 microM) was completely suppressed by additional application of glibenclamide (1 microM). In contrast, glibenclamide (1-10 microM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (> or = microM). 3. Bay K8644 (1 microM) reduced the maximum relaxation produced by ZD6169 (> or =10 microM). 4. In whole-cell configuration, ZD6169 suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 100 microM, shifted the steady-state inactivation curve of the voltage-dependent Ba(2+) currents to the left at a holding potential of -90 mV. 5. In cell-attached configuration, open probability of unitary voltage-dependent Ba(2+) channels (27 pS, 90 mM Ba(2+)) was inhibited by 100 microM ZD6169 and by 10 microM nifedipine. 6. Reverse transcriptase-polymerase chain reaction (RT - PCR) analysis revealed the presence of the transcript of the alpha(1C) subunit of L-type Ca(2+) channels in pig urethra. 7. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of K(ATP) channels at lower concentrations and inhibition of voltage-dependent Ca(2+) channels at higher concentrations (about 10 microM).

Gene expression of SERCA2a and L- and T-type Ca channels during human heart development.

In this study we report, for the first time, on the gene expression of human cardiac SERCA2a, L-type (alpha(1C)) and T-type (alpha(1H)) Ca channels during development, using RNase protection assay, relative quantitative RT-PCR and Western blot. Human hearts during early gestation (8- to 20-wk gestation), neonatal (1- to 4-d-old) and adult (18- to 48-year-old) stages were used. The results show that T-type Ca channel alpha(1H) subunit mRNA decreased and that L-type Ca channel alpha(1C) subunit mRNA increased with development. While the levels of sarcoplasmic reticulum ATPase (SERCA2a) mRNA did not significantly change with development, its protein levels increased with development. In conclusion, SERCA2a, L-type and T-type Ca channel transcripts were detected as early as 8-wk gestation. Defining the profile of Ca handling proteins during development is important to the understanding of excitation-contraction (EC)-coupling of the developing human heart.

Interaction of SNX482 with Domains III and IV Inhibits Activation Gating of α1E (Ca V2.3) Calcium Channels

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type α 1E calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an ∼70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits α 1E calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of α 1E and α 1C subunits indicated that the presence of the domain III and IV of α 1E is a prerequisite for a strong gating inhibition. In contrast, L-type α 1C channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.

C-terminal Fragments of the α1C(CaV1.2) Subunit Associate with and Regulate L-type Calcium Channels Containing C-terminal-truncated α1CSubunits

L-type Ca(2+) channels in native tissues have been found to contain a pore-forming alpha(1) subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminal-truncated alpha(1C) (Ca(V)1.2) subunits, we performed electrophysiological and biochemical experiments. In tsA201 cells expressing either wild type or C-terminal-truncated alpha(1C) subunits in combination with a beta(2a) subunit, truncation of the alpha(1C) subunit by as little as 147 amino acids led to a 10-15-fold increase in currents compared with those obtained from control, full-length alpha(1C) subunits. Dialysis of cells expressing the truncated alpha(1C) subunits with C-terminal fragments applied through the patch pipette reconstituted the inhibition of the channels seen with full-length alpha(1C) subunits. In addition, C-terminal deletion mutants containing a tethered C terminus also exhibited the C-terminal-induced inhibition. Immunoprecipitation assays demonstrated the association of the C-terminal fragments with truncated alpha(1C) subunits. In addition, glutathione S-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the C- terminal fragments of the alpha(1C) subunit can associate with C-terminal-truncated alpha(1C) subunits and inhibit the currents through L-type Ca(2+) channels.

Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum

In the present study, we used immunohistochemistry and Western blot analysis to determine region-specific changes in the distribution of voltage-gated calcium channels (VGCCs) in aged rat cerebellum. Age-dependent changes in the staining intensity of the α 1C and α 1D subunits were prominent in the Purkinje cells, whereas there was no change in the expression of the α 1A and α 1B subunits. In the aged rat, in particular, immunoreactivity for the α 1C subunits were increased in the dendrites as well as in the cell bodies of Purkinje cells. On the other hand, decreases in immunoreactivity for α 1A and α 1D subunits were found in the molecular or granular layers. However, only α 1D subunit immunoreactivity was decreased in the aged cerebellum membrane by Western blot analysis that, while not addressing regional specificity, further confirmed an age-related decrease in α 1D subunit. These age-related changes in α 1D subunit expression might reflect a gradual loss of regulation for L-type Ca 2+ channels in the senescent period. The first demonstration of age-related alterations in VGCC expression may provide useful data for future investigations on aging and neurodegenerative diseases.

Transient down-regulation of L-type Ca(2+) channel and dystrophin expression after balloon injury in rat aortic cells.

Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

Ca 2+ sensors of L-type Ca 2+ channel

Ca 2+-induced inactivation of L-type Ca 2+ is differentially mediated by two C-terminal motifs of the α 1C subunit, L (1572–1587) and K (1599–1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca 2+ sensor and an adjacent Ca 2+-independent tethering site for calmodulin. The Ca 2+ sensor contributes to higher Ca 2+ sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca 2+-dependent current decay, the two-site modulation by Ca 2+ and calmodulin may underlie Ca 2+-induced inactivation of the channel.

Serine Residue in the IIIS5-S6 Linker of the L-type Ca2+ Channel α1C Subunit Is the Critical Determinant of the Action of Dihydropyridine Ca2+Channel Agonists

The dihydropyridine (DHP)-binding site has been identified within L-type Ca(2+) channel alpha(1C) subunit. However, the molecular mechanism underlying modulation of Ca(2+) channel gating by DHPs has not been clarified. To search for novel determinants of high affinity DHP binding, we introduced point mutations in the rat brain Ca(2+) channel alpha(1C) subunit (rbCII or Ca(v)1.2c) based on the comparison of amino acid sequences between rbCII and the ascidian L-type Ca(2+) channel alpha(1) subunit, which is insensitive to DHPs. The alpha(1C) mutants (S1115A, S1146A, and A1420S) and rbCII were transiently expressed in BHK6 cells with beta(1a) and alpha(2)/delta subunits. The mutation did not affect the electrophysiological properties of the Ca(2+) channel, or the voltage- and concentration-dependent block of Ca(2+) channel currents produced by diltiazem and verapamil. However, the S1115A channel was significantly less sensitive to DHP antagonists. Interestingly, in the S1115A channel, DHP agonists failed to enhance whole-cell Ca(2+) channel currents and the prolongation of mean open time, as well as the increment of NP(o). Responsiveness to the non-DHP agonist FPL-64176 was also markedly reduced in the S1115A channel. When S1115 was replaced by other amino acids (S1115D, S1115T, or S1115V), only S1115T was slightly sensitive to S-(-)-Bay K 8644. These results indicate that the hydroxyl group of Ser(1115) in IIIS5-S6 linker of the L-type Ca(2+) channel alpha(1C) subunit plays a critical role in DHP binding and in the action of DHP Ca(2+) channel agonists.

Modulation of L-type Ca2+ Channels by Gβγ and Calmodulin via Interactions with N and C Termini of α1C

Neuronal voltage-dependent Ca(2+) channels of the N (alpha(1B)) and P/Q (alpha(1A)) type are inhibited by neurotransmitters that activate G(i/o) G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gbetagamma subunit of G proteins to the channel. Since cardiac and neuronal L-type (alpha(1C)) voltage-dependent Ca(2+) channels are not modulated in this way, they are presumed to lack interaction with Gbetagamma. However, here we demonstrate that both Gbetagamma and calmodulin directly bind to cytosolic N and C termini of the alpha(1C) subunit. Coexpression of Gbetagamma reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca(2+), and is eliminated by EGTA. The N and C termini of alpha(1C) appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gbetagamma. Deletion of the N terminus profoundly impairs the Ca(2+)/calmodulin-dependent inactivation. We propose that Gbetagamma and calmodulin regulate the L-type Ca(2+) channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of alpha(1C).

Protein Phosphatase 2A Is Associated with Class C L-type Calcium Channels (Cav1.2) and Antagonizes Channel Phosphorylation by cAMP-dependent Protein Kinase

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.