Ca2+- and voltage-dependent inactivation of the expressed L-type Ca(v)1.2 calcium channel

Ca2+-dependent regulation of the ion current through the alpha1Cbeta2aalpha2delta-1 (L-type) calcium channel transiently expressed in HEK 293 cells was investigated using whole cell patch clamp method. Ca2+ or Na+ ions were used as a charge carrier. Intracellular Ca2+ was either buffered by 10 mM EGTA or 200 microM Ca2+ was added into non-buffered intracellular solution. Free intracellular Ca2+ inactivated permanently about 80% of the L-type calcium current. The L-type calcium channel inactivated during a depolarizing pulse with two time constants, tau(fast) and tau(slow). Free intracellular calcium accelerated both time constants. Effect on the tau(slow) was more pronounced. About 80% of the channel inactivation during brief depolarizing pulse could be attributed to a Ca2+-dependent mechanism and 20% to a voltage-dependent mechanism. When Na+ ions were used as a charge carrier, the L-type current still inactivated with two time constants that were 10 times slower and were virtually voltage-independent. Ca2+ ions stabilized the inactivated state of the channel in a concentration-dependent manner.     

alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation

Smooth muscle exhibitsmechanosensitivity independent of neural input, suggesting thatmechanosensitive pathways reside within smooth muscle cells. The nativeL-type calcium current recorded from human intestinal smooth muscle ismodulated by stretch. To define mechanosensitive mechanisms involved inthe regulation of smooth muscle calcium entry, we cloned the_1C L-type calcium channel subunit (Ca_V1.2)from human intestinal smooth muscle and expressed the channel in aheterologous system. This channel subunit retained mechanosensitivitywhen expressed alone or coexpressed with a ₂ calciumchannel subunit in HEK-293 or Chinese hamster ovary cells. Theheterologously expressed human cardiac _1C splice formalso demonstrated mechanosensitivity. Inhibition of kinase signalingdid not affect mechanosensitivity of the native channel. Truncation of the _1C COOH terminus, which containsan inhibitory domain and a proline-rich domain thought to mediatemechanosensitive signaling from integrins, did not disruptmechanosensitivity of the expressed channel. These data demonstratemechanical regulation of calcium entry through molecularly identifiedL-type calcium channels in mammalian cells and suggest that themechanosensitivity resides within the pore forming_1C-subunit.

     

Role of the C terminus of the alpha 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels

We have previously demonstrated that formation of a complex between L-type calcium (Ca(2+)) channel alpha(1C) (Ca(V)1.2) and beta subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the alpha(1C) subunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the alpha(1C) subunit, a domain consisting of amino acid residues 1623-1666 ("targeting domain") in the C terminus of the alpha(1C) subunit has been identified to be important for correct targeting of L-type Ca(2+) channel complexes to the plasma membrane. Although cells expressing the wild-type alpha(1C) and beta(2a) subunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the alpha(1C) subunit that lack the targeting domain with the beta(2a) subunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the alpha(1C) subunit that were downstream of residues 1623-1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the alpha(1C) subunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the alpha(1C) subunit was required for proper plasma membrane localization of the L-type Ca(2+) channels.     

Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels

Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.     

The NH2 terminus of RCK1 domain regulates Ca2+-dependent BK(Ca) channel gating

Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Fiber-FISH analysis of the 3'-terminal region of the human L-type Ca2+ channel alpha 1C subunit gene

Human L-type Ca2+ channel alpha 1C subunit gene (CACNL1A1) maps to the distal region of chromosome 12p13, and is composed of approximately 50 exons spanning over 150 kb of the human genome as estimated by restriction map analysis. However, the structure and the total length of the 3'-end of the gene is not clear because the size of several big introns remains unknown. Here the fiber-FISH technique was used to determine the relative order and size of eight partial genomic DNA clones from the central and 3'-terminal regions of CACNL1A1. The total physical distance of this region, including the size and gap distances between the clones were re-estimated. The results show that the physical order of the tested clones was 5'-g14-5 > g12-2 > g10-8 > g4-5 > g16-7 > g8-3 > g12-5 > g6-20-3'. Their individual sizes vary between 6.7 and 21.9 kb. Clones g6-20 and g12-5, both containing repetitive exon 45/46-like element, were found to be located within 59.1 kb downstream of g8-3 containing earlier identified polyadenylation site, i.e. 229.5 kb away from clone g14-5 (exons 10, 11). The possible implications of this structural complexity is discussed.     

A sequence in the carboxy-terminus of the alpha(1C) subunit important for targeting, conductance and open probability of L-type Ca(2+) channels

The role of the 80-amino acid motif 1572-1651 in the C-terminal tail of alpha(1C) Ca(2+) channel subunits was studied by comparing properties of the conventional alpha(1C,77) channel expressed in HEK-tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572-1651 in alpha(1C,77) with 81 non-identical residues leading to alpha(1C,86) impaired membrane targeting and cluster formation of the channel. Similar to alpha(1C, 86), substitution of its 1572-1598 (alpha(1C,77L)) or 1595-1652 (alpha(1C,77K)) segments into the alpha(1C,77) channel yielded single-channel Ba(2+) currents with increased inactivation, reduced open probability and unitary conductance, when compared to the alpha(1C,77) channel. Thus, the C-terminal sequence 1572-1651 of the alpha(1C) subunit is important for membrane targeting, permeation and open probability of L-type Ca(2+) channels.     

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.     

Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca(v)1.2) in a cooperative manner

Syntaxin 1A (Sx1A) modifies the activity of voltage-gated Ca2+ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca2+ channel, Cav1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits alpha11.2/alpha2delta1/beta2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca2+ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca2+ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca2+ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.     

L-type Ca2+ channel facilitation mediated by phosphorylation of the beta subunit by CaMKII

L-type Ca(2+) channels (LTCCs) are major entry points for Ca(2+) in many cells. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is associated with cardiac LTCC complexes and increases channel open probability (P(O)) to dynamically increase Ca(2+) current (I(Ca)) and augment cellular Ca(2+) signaling by a process called facilitation. However, the critical molecular mechanisms for CaMKII localization to LTCCs and I(Ca) facilitation in cardiomyocytes have not been defined. We show CaMKII binds to the LTCC beta(2a) subunit and preferentially phosphorylates Thr498 in beta(2a). Mutation of Thr498 to Ala (T498A) in beta(2a) prevents CaMKII-mediated increases in the P(O) of recombinant LTCCs. Moreover, expression of beta(2a)(T498A) in adult cardiomyocytes ablates CaMKII-mediated I(Ca) facilitation, demonstrating that phosphorylation of beta(2a) at Thr498 modulates native calcium channels. These findings reveal a molecular mechanism for targeting CaMKII to LTCCs and facilitating I(Ca) that may modulate Ca(2+) entry in diverse cell types coexpressing CaMKII and the beta(2a) subunit.     

Serine residue in the IIIS5-S6 linker of the L-type Ca2+ channel alpha 1C 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.     

The N terminus of the cardiac L-type Ca(2+) channel alpha(1C) subunit. The initial segment is ubiquitous and crucial for protein kinase C modulation, but is not directly phosphorylated.

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca(2+) channel alpha(1C) subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901-17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of alpha(1C) leave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate the presence of alpha(1C) protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of alpha(1C) expressed in Xenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathione S-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.     

Regulation of cardiac L-type Ca2+ channel by coexpression of G(alpha s) in Xenopus oocytes

Activation of G(alpha s) via beta-adrenergic receptors enhances the activity of cardiac voltage-dependent Ca2+ channels of the L-type, mainly via protein kinase A (PKA)-dependent phosphorylation. Contribution of a PKA-independent effect of G(alpha s) has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous G(alpha s) reduced, whereas coexpression of G(alpha s) enhanced, currents via expressed cardiac L-type channels, independently of the presence of the auxiliary subunits alpha2/delta or beta2A. Coexpression of G(alpha s) did not increase the amount of alpha1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed beta2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP-dependent decrease was observed. We conclude that coexpression of G(alpha s), but not its acute activation via beta-adrenergic receptors, enhances the activity of the cardiac L-type Ca2+ channel via a PKA-independent effect on the alpha1C subunit.     

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death

Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.     

Impaired Ca2+ homeostasis is associated with atrial fibrillation in the alpha1D L-type Ca2+ channel KO mouse

The novel alpha1D Ca2+ channel together with alpha1C Ca2+ channel contribute to the L-type Ca2+ current (I(Ca-L)) in the mouse supraventricular tissue. However, its functional role in the heart is just emerging. We used the alpha1D gene knockout (KO) mouse to investigate the electrophysiological features, the relative contribution of the alpha1D Ca2+ channel to the global I(Ca-L), the intracellular Ca2+ transient, the Ca2+ handling by the sarcoplasmic reticulum (SR), and the inducibility of atrial fibrillation (AF). In vivo and ex vivo ECG recordings from alpha1D KO mice demonstrated significant sinus bradycardia, atrioventricular block, and vulnerability to AF. The wild-type mice showed no ECG abnormalities and no AF. Patch-clamp recordings from isolated alpha1D KO atrial myocytes revealed a significant reduction of I(Ca-L) (24.5%; P < 0.05). However, there were no changes in other currents such as I(Na), I(Ca-T), I(K), I(f), and I(to) and no changes in alpha1C mRNA levels of alpha1D KO atria. Fura 2-loaded atrial myocytes showed reduced intracellular Ca2+ transient (approximately 40%; P < 0.05) and rapid caffeine application caused a 17% reduction of the SR Ca2+ content (P < 0.05) and a 28% reduction (P < 0.05) of fractional SR Ca2+ release in alpha1D KO atria. In conclusion, genetic deletion of alpha1D Ca2+ channel in mice results in atrial electrocardiographic abnormalities and AF vulnerability. The electrical abnormalities in the alpha1D KO mice were associated with a decrease in the total I(Ca-L) density, a reduction in intracellular Ca2+ transient, and impaired intracellular Ca2+ handling. These findings provide new insights into the mechanism leading to atrial electrical dysfunction in the alpha1D KO mice.     

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.