Voltage-gated Calcium Channel Cav1 Research Articles

Differential Effects of Crambescins and Crambescidin 816 in Voltage-Gated Sodium, Potassium and Calcium Channels in Neurons

Crambescins and crambescidins are two families of guanidine alkaloids from the marine sponge Crambe crambe. Although very little information about their biological effect has been reported, it is known that crambescidin 816 (Cramb816) blocks calcium channels in a neuroblastoma X glioma cell line. Taking this into account, and the fact that ion channels are frequent targets for natural toxins, we examined the effect of Cramb816 and three compounds from the crambescin family, norcrambescin A2 (NcrambA2), crambescin A2 (CrambA2), and crambescin C1 (CrambC1), in the main voltage-dependent ion channels in neurons: sodium, potassium, and calcium channels. Electrophysiological recordings of voltage gated sodium, potassium, and calcium currents, in the presence of these guanidine alkaloids, were performed in cortical neurons from embryonic mice. Different effects were discovered: crambescins inhibited K(+) currents with the following potency: NcrambA2 > CrambC1 > CrambA2, while Cramb816 lacked an effect. Only CrambC1 and Cramb816 partially blocked Na(+) total current. However, Cramb816 partially blocked Ca(2+) , while NcrambA2 did not. Since the blocking effect of Cramb816 on calcium currents has not been previously reported in detail, we further pharmacologically isolated the two main fractions of HVA Ca(2+) channels in neurons and investigated the Cramb816 effect on them. Here, we revealed that Cav1 or L-type calcium channels are the main target for Cramb816. These two families of guanidine alkaloids clearly showed a structure-activity relationship with the crambescins acting on voltage-gated potassium channels, while Cramb816 blocks the voltage-gated calcium channel Cav1 with higher potency than nifedipine. The novel evidence that Cramb816 partially blocked CaV and NaV channels in neurons suggests that this compound might be involved in decreasing the neurotransmitter release and synaptic transmission in the central nervous system. The findings presented here provide the first detailed approach on the different effects of crambescin and crambescidin compounds in voltage-gated sodium, potassium, and calcium channels in neurons and thus provide a basis for future studies.

Competition between Calcium Sensors on the Voltage-Gated Calcium Channel Cav1.2

Voltage-gated calcium channel (CaV) activity is regulated by calcium sensors including calmodulin (CaM) and calcium-binding protein 1 (CaBP1). CaBP1 inhibits CaM-mediated calcium-dependent inactivation (CDI). We investigated the origins of functionally important differences between CaM and CaBP1 by creating a number of CaM/CaBP1 chimeras which suggest that both calcium sensors use their C-lobes for high affinity binding to the pore-forming CaV alpha-subunit, while their N-terminal lobe are responsible for the stark functional differences of the calcium sensors. CaBP1 and CaM are thought to modulate CaV function by competing for binding to the CaV C-terminal IQ-domain, but this assumption has never been tested directly. By determining CaV1.2 CDI in Xenopus oocytes under conditions with different ratios of CaM and CaBP1, we demonstrate direct competition between both calcium sensors for their CaV1.2 binding site. In order to extend our analysis of CaBP1/CaM competition we used isothermal titration calorimetry to determine the affinity of both CaM and CaBP1 in both calcium-bound and apo-states for the IQ domain, suggesting that competition occurs mainly in the apo-state. Overall, our data provide a framework for understanding how CaBP1 and CaM differentially regulate CDI on CaV1.2.

Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity

An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃]-dependent Ca(2+) signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P₃ production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P₃ was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 µM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-α1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 µM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 µM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.

Single-Channel Mechanism of Modulation of Calcium-Dependent Inactivation of the Voltage-Gated Calcium Channel Cav1.3 by its C-Terminus

Background: CaV1.3 is a L-type calcium channel pore subunit showing calcium-dependent inactivation (CDI). The full-length CaV1.3 isoform CaV1.342 contains a C-terminal modulator (CTM) domain that attenuates calcium-dependent inactivation at the whole-cell current level. CaV1.3 splice variants lack the CTM domain completely (CaV1.342A) or partially (CaV1.343S).Aim of the study: To analyse the role of the CTM domain for single-channel gating of CaV1.342, CaV1.342A and CaV1.343S in detail.Methods: We recorded single-channel calcium and barium currents carried by either CaV1.342, CaV1.342A and CaV1.343S plus auxiliary subunits expressed in HEK293 cells. Single-channel CDI was analysed according to Josephson et al. (J Physiol 2010;588:213). By Markov modelling transition rates between channel states were obtained.Results: As expected single-channel barium currents showed little inactivation during 150ms test pulses. Single-channel activity with barium was different with CaV1.342A being most and CaV1.342 least active (Bock et al., J Biol Chem 2011; 286:42736). This difference is due to CTM-dependent changes in the closed-to-open and open-to-closed transition rates across all test potentials examined (−20 to 0 mV). With calcium as charge carrier the CaV1.3 variants CaV1.342A and CaV1.343S lacking the CTM domain allowed for more pronounced CDI compared to full-length CaV1.342. Plotting the ratio of mean open times during late (MOT2) and early (MOT1) period of a depolarizing pulse against cumulative amount of calcium ions passing the channel reveals that a shortening of mean open times underlies CDI of CaV1.342A and CaV1.343S. The efficacy of calcium ions to induce this readout parameter of CDI was higher with the isoforms (partly) lacking CTM.Conclusion: CTM reduces the sensitivity of single CaV1.3 to CDI. Splice variants affecting the structure of CTM thus will influence the physiological function of e.g. cardiac pacemaker cells or neurons.

Aminopiperidine Sulfonamide Cav2.2 Channel Inhibitors for the Treatment of Chronic Pain

The voltage-gated calcium channel Ca(v)2.2 (N-type calcium channel) is a critical regulator of synaptic transmission and has emerged as an attractive target for the treatment of chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors of Ca(v)2.2. In particular, 19 is an inhibitor of Ca(v)2.2 that is selective over cardiac ion channels, with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Ca(v)2.2 gene-deleted mice. The discovery of metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also suggests specific structural liabilities of this class of compounds that must be addressed.

Blockade of voltage-gated calcium channel Cav1.2 and α1-adrenoceptors increases vertebral artery blood flow induced by the antivertigo agent difenidol

Difenidol (1,1-diphenyl-4-piperidino-1-butanol hydrochloride) is an effective drug for the treatment of vertigo and dizziness. This drug is known to improve the blood flow in vertebral arteries, though the precise mechanism underlying this action remains unclear. In the present study, we investigated the effect of difenidol on voltage-gated calcium channel Cav1.2 and α1-adrenoceptor subtypes that regulate the intracellular calcium concentration ([Ca2+]i), as well as their possible involvement in the action of difenidol on vertebral artery relaxation and blood flow in dogs. In vitro binding assays demonstrated that difenidol at micromolar concentrations bound to the α1A-, α1B- and α1D-adrenoceptor subtypes. Difenidol inhibited the phenylephrine-induced increase in [Ca2+]i in Chinese hamster ovary cells expressing human α1A-, α1B- or α1D-adrenoceptor subtypes with similar IC50 values in the low micromolar range. In an electrophysiological assay, difenidol inhibited L-type calcium channel (Cav1.2 subunit). In dogs, i.v. difenidol preferentially enhanced vertebral over femoral arterial blood flow. Phenylephrine and potassium induced contraction of dog vertebral arterial rings, and difenidol inhibited this action. Inhibition of phenylephrine-induced contraction by difenidol was mimicked by the α1-adrenoceptor antagonist phentolamine, the α1A-adrenoceptor antagonist RS 17,053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine hydrochloride) and the α1D-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride). In addition, the L-type calcium channel blocker nifedipine, like difenidol, attenuated the potassium-induced contraction. These findings suggest that the difenidol-induced increase in vertebral arterial blood flow may be due to vascular relaxation mediated by mixed blocking actions at α1-adrenoceptors and voltage-gated calcium channel Cav1.2.

Analysis of Competition between CaBP1 and Calmodulin on the Voltage-Gated Calcium Channel Cav1.2

Voltage-gated calcium channel (CaV) activity is regulated by calcium sensors including calmodulin (CaM) and calcium-binding protein 1 (CaBP1). CaBP1 inhibits CaM-mediated calcium-dependent inactivation (CDI). We investigated the origins of functionally important differences between CaM and CaBP1 by creating a number of CaM/CaBP1 chimeras. The exchange of C-terminal lobes between CaM and CaBP1 is accompanied by few functional changes, suggesting CaBP1 and CaM C-lobe can substitute for each other. In contrast, we identified the linker and the N-terminal lobe of CaBP1 as elements that set it functionally apart from CaM. CaBP1 and CaM are thought to modulate CaV function by competing for binding to the CaV C-terminal IQ-domain, but this assumption has never been tested directly. By determining CaV1.2 CDI under conditions with different ratios of CaM and CaBP1, we demonstrate direct competition between CaBP1 and CaM for its CaV1.2 binding site. In order to extend our analysis of CaBP1/CaM competition we used isothermal titration calorimetry to determine the affinity of both CaM and CaBP1 in both calcium-bound and apo-states for the IQ domain, suggesting that competition occurs mainly in the apo-state. Analysis of an IQ domain mutant shows unexpected differences between CaM and CaBP1 in IQ domain binding, suggesting a possible mechanism for their different functional effects on CaV1.2 inactivation. Overall, the data reveal those parts of CaBP1 that set it functionally apart from CaM and provide a framework for understanding how CaBP1 and CaM differentially regulate CDI on CaV1.2.

Molecular Mechanism of Enhancement of CaV1.2 Channels by β2-Adrenergic Receptors

In neurons, β2-adrenergic (β2AR) enhancement of the activity of L-type voltage-gated calcium channels (Cav1.2) is important for certain forms of synaptic plasticity. The mechanism of regulation is thought to involve phosphorylation by protein kinase A (PKA) of residues in the C-terminus of Cav1.2. A PKA-anchoring protein (AKAP) is required for efficient phosphorylation and enhancement of Cav1.2 channel activity. Neuronally-localized AKAP79, unlike all other AKAPs, binds the Ca2+/calmodulin-activated phosphatase calcineurin (CaN), and thus anchors CaN along with PKA within the Cav1.2-β2AR complex. To study β2AR enhancement of Cav1.2 channel activity, we have used whole-cell patch-clamp to voltage-clamp currents from tsA-201 cells transfected with constructs for full-length Cav1.2, the channel's accessory subunits (β2b, α2δ), AKAP79 and the β2AR. With this system, we have found that β2AR agonist (isoproterenol, Iso) can enhance Ba2+ current ∼25%. Enhancement by Iso was nearly absent (∼5%) when current was carried by Ca2+. Candidates that could support Ca2+-dependent suppression of enhancement include CaN and the Ca2+/calmodulin-dependent kinase CaMKII, which is also scaffolded to the channel. Sites phosphorylated by PKA and suspected of involvement in enhancement include residues S1700, T1704 and S1928 in the CaV1.2 C-terminus. Substitution of alanine at any of these three sites of phosphorylation by PKA abolished enhancement by Iso. To further investigate the mechanism of enhancement, we used the ratio of Ba2+ tail current (−60 mV) to gating charge at reversal as an index of the efficiency of channel opening (Itail/Qgate coupling efficiency). For wild-type (WT) channels, coupling efficiency was increased by Iso. Phosphorylation site mutations (S1700, S1928, S1700+T1704) decreased coupling efficiency in the absence of Iso, and abolished the Iso-induced increase in coupling efficiency observed for WT channels, consistent with the inability of agonist to enhance current carried by the mutants.

A key role of TRPC channels in the regulation of electromechanical activity of the developing heart

It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart. TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the α1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first- and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity. These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

Reply to Eisner et al.: CaMKII phosphorylation of RyR2 increases cardiac contractility

The ryanodine receptor/calcium-release channel (RyR2) on the sarcoplasmic reticulum (SR) is the source of Ca2+ required for myocardial excitation–contraction (EC) coupling. During stress (i.e., exercise), contractility of the cardiac muscle is increased largely because of phosphorylation and activation of key proteins that regulate SR Ca2+ release. These include the voltage-gated calcium channel (Cav1.2) on the plasma membrane through which Ca2+ enters the cardiomyocyte, the sarco/endoplasmic reticulum calcium ATPase (SERCA2a)/phospholamban complex that pumps Ca2+ into the SR, and the RyR2 channel that releases Ca2+ from the SR, all of which are activated by phosphorylation.

Conformational Changes Induced in Voltage-gated Calcium Channel Cav1.2 by BayK 8644 or FPL64176 Modify the Kinetics of Secretion Independently of Ca2+ Influx

The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca(2+) transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 microm BayK or 0.5 microm FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca(2+) and Ba(2+) or the impermeable charge carrier La(3+). These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca(2+)-impermeable channel made by replacing the wild type alpha(1)1.2 subunit was replaced with the mutant alpha(1)1.2/L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltage-gated Ca(2+) channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.

Structural and Functional Comparison Between the Effects of CaBP1 and Calmodulin on the Voltage-Gated Calcium Channel Cav1.2

Calcium-dependent feedback modulation is central to voltage-gated calcium channel (CaV) function. The bilobed EF-hand containing calcium-binding protein 1 (CaBP1) is thought to modulate CaV function by competing with the homologous calmodulin (CaM) for binding to the CaV C-terminal IQ-domain. In CaV1.2, CaBP1 inhibits calcium-dependent inactivation (CDI) of CaV1.2 and introduces calcium-dependent facilitation (CDF). We investigated the origins of functionally important differences between CaM and CaBP1 by creating a number of CaM/CaBP1 chimeras and found a clear division of function between the various elements. Determination of the CaBP1 X-ray structure revealed an interaction site between linker and one of the lobes that is required for inhibition of CaV1.2 CDI. Using titration calorimetry, we found that, similar to CaM, the CaBP1 C-terminal lobe is responsible for high-affinity interaction with the IQ-domain. CaM requires functional C-terminal lobe EF-hands for supporting CDI. In contrast, we found that CaBP1 does not require functional EF-hands for inhibition of CDI. CaBP1-mediated CDF however requires all EF-hands to be functional and also has different requirements from CaM-mediated CDF, suggesting that these are two distinct processes. Overall, the data reveal those parts of CaBP1 that set it functionally apart from CaM and provide a framework for understanding how CaBP1 and CaM regulate CDI and CDF on CaV1.2.

Impaired long-term potentiation and enhanced neuronal excitability in the amygdala of Ca V1.3 knockout mice

Previously, we demonstrated that mice in which the gene for the L-type voltage-gated calcium channel Ca V1.3 is deleted (Ca V1.3 knockout mice) exhibit an impaired ability to consolidate contextually-conditioned fear. Given that this form of Pavlovian fear conditioning is critically dependent on the basolateral complex of the amygdala (BLA), we were interested in the mechanisms by which Ca V1.3 contributes to BLA neurophysiology. In the present study, we used in vitro amygdala slices prepared from Ca V1.3 knockout mice and wild-type littermates to explore the role of Ca V1.3 in long-term potentiation (LTP) and intrinsic neuronal excitability in the BLA. We found that LTP in the lateral nucleus (LA) of the BLA, induced by high-frequency stimulation of the external capsule, was significantly reduced in Ca V1.3 knockout mice. Additionally, we found that BLA principal neurons from Ca V1.3 knockout mice were hyperexcitable, exhibiting significant increases in firing rates and decreased interspike intervals in response to prolonged somatic depolarization. This aberrant increase in neuronal excitability appears to be at least in part due to a concomitant reduction in the slow component of the post-burst afterhyperpolarization. Together, these results demonstrate altered neuronal function in the BLA of Ca V1.3 knockout mice which may account for the impaired ability of these mice to consolidate contextually-conditioned fear.

G010 Regulation of voltage-dependent calcium channels by Nedd4-1

Calcium entry into excitable cells can be regulated by controlling both the activity of calcium channels and the amount of channels available at the plasma membrane. Little is known about their internalisation and degradation. One post-translational modification shown to be involved in membrane protein internalisation and subsequent degradation is the attachment of ubiquitin moieties by ubiquitin-ligases. Previously, it has been shown that cardiac ion channels such as Nav1.5 and KCNQ1 are down-regulated by ubiquitin-ligases of the Nedd4 family. These regulations involve the interaction between the PY motif of the target channels and the WW motif of the ubiquitin-ligases. Despite the absence of such PY motif in the cardiac voltage-gated calcium channel Cav1.2, we investigated whether this channel could by regulated in the same manner than other cardiac channels as previously described. We co-expressed, in HEK293 cells, L-type calcium channels and its two regulatory subunits Cavbeta and Cavalpha2delta1 together with ubiquitin-ligases, and examined by voltage-clamp whole-cell recordings the calcium current. We next determined by western blot and surface biotinylation assays the availability of the different subunits of calcium channels in total HEK293 lysates, and at the cell surface. Levels of ubiquitylation of the different subunits were assessed by pull-down GST-S5A and immunoprecipitation of Cav1.2 and its subunits. We found that co-expressing the ubiquitin-ligase Nedd4-1 significantly reduced Cav currents, and decreased Cavalpha and its subunit protein levels. This effect was Nedd4-1 specific since none of the other members of the Nedd4 family we tested produced a similar effect. We also found that the effect of Nedd4-1 was dependent on the co-expression of the Cavbeta subunit. No Nedd4-1-dependent increase in ubiquitylation of the Cavalpha protein was found; and unexpectedly, the two regulating subunits Cavbeta and Cavalpha2delta1 were detected to be deubiquitylated upon Nedd4-1 co-expression. Our data suggest that Nedd4-1 regulates the expression of Cav channels and its subunits by Nedd4-1 via an indirect mechanism constituting a new regulatory pathway to be determined. Further experiments will focus on the role of adrenergic receptors known to be ubiquitylated by Nedd4 and to bind to Cav1.2.

Direct Interaction of Otoferlin with Syntaxin 1A, SNAP-25, and the L-type Voltage-gated Calcium Channel CaV1.3

The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca2+-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca2+-dependent manner (with optimal 61 microm free Ca2+ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca2+, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (Kd = 1.7 x 10(-5) m) than for the C2F interaction (Kd = 2.6 x 10(-9) m). In contrast, it was the otoferlin C2D domain that bound the Cav1.3 II-III loop, in a Ca2+-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca2+ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Cav1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function.

Angiotensin II causes endothelial-dependent increase in expression of Ca V1.2 protein in cultured arteries

Examination was made of the direct vascular effects of the hypertension-inducing pressor hormone angiotensin II on expression and activity of the voltage-gated calcium channel Ca V1.2. Freshly dissected rat superior mesenteric artery beds were maintained in organ culture unpressurized for 24 h in the presence or absence of angiotensin II. Relative to controls, angiotensin II increased Ca V1.2 protein expression and tension-inducing activity but not Ca V1.2 message. The increase in Ca V1.2 protein expression by angiotensin II was abrogated by damaging the endothelium. Thus, the endothelium is involved in regulating Ca V1.2 expression in the vascular wall.

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional “Ca2+ nanodomain” where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.

Apo calmodulin binding to the L-type voltage-gated calcium channel Ca v1.2 IQ peptide

The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic recticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645–1668) housed within the cytoplasmic domain of the LTCC Ca v1.2 subunit has been shown to bind both calcium-loaded (Ca 2+CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca 2+CaM can bind to the intact channel.

Channel to the Nucleus?

Gomez-Ospina et al . provide evidence for an intriguing mechanism whereby voltage-dependent calcium channels modulate gene transcription. Calcium entry through voltage-gated channels in the plasma membrane provides a link between electrical activity and changes in gene expression. Gomez-Ospina et al . found that, whereas an antibody that recognized the full-length Ca v 1.2 calcium channel localized to membrane and cytosolic fractions of rat brain cortex, an antibody that recognized the C-terminal fragment, which is proteolytically cleaved, appeared in the nucleus. The C-terminal fragment was abundant in nuclei of GABAergic neurons; moreover, nuclear fluorescence was apparent in neurons or glioblastoma cells expressing a construct in which the Ca v 1.2 C terminus was fluorescently labeled. Treatments aimed at lowering cytoplasmic calcium increased nuclear abundance of the Ca v 1.2 fragment, whereas treatments that increase intracellular calcium decreased it. The Ca v 1.2 fragment immunoprecipitated with p54(nrb)/NonO, a nuclear protein, and, when recruited to the promoter with a heterologous DNA binding domain, transcriptionally activated a gene reporter. Overexpression of the fragment, which the authors called calcium channel associated transcription regulator (CCAT), led to an increase in the expression of some endogenous genes and a decrease in the expression of others. CCAT overexpression increased the abundance of mRNA encoding the gap junction protein Cx31.1 and transcriptionally activated a reporter containing the Cx31.1 promoter; chromatin immunoprecipitation analysis indicated that it associated with the Cx31.1 promoter. Furthermore, decreasing the abundance of endogenous nuclear CCAT either by depolarization or with short hairpin RNA led to a decrease in transcription of the Cx31.1 reporter. Thus, the authors conclude that the proteolytically cleaved C terminus of Ca v 1.2 translocates to the nucleus to act as a transcription factor. N. Gomez-Ospina, F. Tsuruta, O. Barreto-Chang, L. Hu, R. Dolmetsch, The C terminus of the L-type voltage-gated calcium channel Ca V 1.2 encodes a transcription factor. Cell 127 , 591-606 (2006). [PubMed]

BK Ca -Cav Channel Complexes Mediate Rapid and Localized Ca 2+ -Activated K + Signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.