Inactivation Of L-type Calcium Channel Research Articles

Local Control Model of a Human Ventricular Myocyte: An Exploration of Frequency-Dependent Changes and Calcium Sparks.

Calcium (Ca2+) sparks are the elementary events of excitation-contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca2+ ([Ca2+]i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca2+ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca2+]myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted "nonconducting" form of Ca2+-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca2+ fractional release was lower due to the smaller difference between diastolic and systolic [Ca2+]SR. Restitution analysis through the S1S2 protocol and increased LCC Ca2+-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca2+ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca2+ release events and ion channel responses throughout various stimuli.

STAC proteins inhibit the voltage dependent inactivation of l-type calcium channels.

The inactivation of voltage-gated calcium channels is governed by two distinct processes: voltage dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The STAC family of adaptor proteins inhibits the CDI of L-type calcium channels (LTCC). In fact, all LTCC (CaV1.2, CaV1.3 and CaV1.4) showed reduced CDI in the presence of any STAC isoform. However, whether STAC proteins also modulate the CDI of the skeletal muscle channel CaV1.1 was initially not investigated, as CaV1.1 requires STAC3 for its functional expression. Interestingly, we could recently demonstrate that STAC3 does not inhibit the CDI, but the VDI of CaV1.1. Therefore, we hypothesized that STAC proteins inhibit also the VDI of LTCC. Indeed, when co-expressed in HEK cells, all STAC isoforms reduced the VDI of CaV1.2 and CaV1.3 currents. As for CDI, also the VDI inhibition is specific for LTCC, as the VDI of CaV2.1 and CaV2.3 is not affected by STAC proteins. As the predominant contribution to VDI is known to arise from the I-II loop interaction with the CaVβ subunit, and that STAC proteins were shown to interact with distinct cytoplasmic domains (II-III loop and C-terminus), we hypothesized that STAC proteins and membrane tethered β subunits utilize different mechanism to inhibit VDI. Indeed, co-expression of STAC3 with CaV1.3/β2a revealed additional deceleration of VDI in HEK cells. Ongoing experiments, involving STAC and channels mutants and chimeras, will reveal the molecular determinants of the VDI inhibition by STAC proteins.

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca2+-sensingprotein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Cav1.2 Ca2+/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca2+-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Cav1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Cav1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.

STAC3 determines the slow activation kinetics of CaV 1.1 currents and inhibits its voltage-dependent inactivation.

The skeletal muscle CaV 1.1 channel functions as the voltage-sensor of excitation-contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both CaV 1.1 functional expression and EC coupling. Interestingly, STAC proteins were also reported to inhibit calcium-dependent inactivation (CDI) of L-typecalcium channels (LTCC), an important negative feedback mechanism in calcium signaling. The same could not be demonstrated for CaV 1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, CaV 1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in CaV 1.1 currents reflects the inhibitory effect of STAC3. Here, we inserted a triple mutation in the linker region of STAC3 (ETLAAA), as the analogous mutation abolished the inhibitory effect of STAC2 on CDI of CaV 1.3 currents. When coexpressed in CaV 1.1/STAC3 double knockout myotubes, the mutant STAC3-ETLAAA failed to colocalize with CaV 1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch-clamp and calcium recording experiments revealed that STAC3-ETLAAA supports CaV 1.1 functional expression and EC coupling, although at a reduced extent compared to wild-type STAC3. Importantly, STAC3-ETLAAA coexpression dramatically accelerated the kinetics of activation and inactivation of CaV 1.1 currents, suggesting that STAC3 determines the slow CaV 1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of CaV 1.1 currents, we performed patch-clamp recordings using calcium and barium as charge carriers in HEK cells. While CaV 1.1 displayed negligible CDI with STAC3, this did not increase in the presence of STAC3-ETLAAA. On the contrary, our data demonstrate that STAC3 specifically inhibits the voltage-dependent inactivation (VDI) of CaV 1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of CaV 1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC.

STAC proteins inhibit calcium- and voltage-dependent inactivation of L-type calcium channels

STAC proteins inhibit calcium- and voltage-dependent inactivation of L-type calcium channels

STAC proteins inhibit calcium and voltage dependent inactivation of L-type calcium channels

Upon depolarization, voltage-gated calcium channels (VGCC) open but then undergo inactivation. Two processes govern VGCC inactivation: calcium-dependent inactivation (CDI) and voltage-dependent inactivation (VDI). Recently, the members of the STAC family of adaptor proteins have been shown to inhibit CDI of L-type calcium channels, by binding to the C-terminal domain. Here, we demonstrate that STAC proteins also inhibit the VDI of CaV1.2 and CaV1.3. Remarkably, using a STAC mutant, we could demonstrate that the inhibition of VDI relies on the same interaction, between STAC and the calcium channel, as CDI.

Capsaicin Causes Vasorelaxation of Rat Aorta through Blocking of L-type Ca2+ Channels and Activation of CB1 Receptors.

The aim of this work was to determine whether Capsaicin may exert a vascular regulation through the activation of CB1 and/or CB2 receptors causing vasorelaxation in the rat aorta. Our results show the location of TRPV1 mainly in the endothelial and smooth muscle cells membrane. Nevertheless, Capsaicin caused vasorelaxation of this artery through a mechanism independent of TRPV1, since the specific antagonists Capsazepine and SB-366791 did not block the effect of Capsaicin. Because the significant expression of CB1 and CB2 receptors has been previously reported in the rat aorta, we used antagonists for these two receptors prior to the addition of Capsaicin. In these experiments, we found that the inhibition of CB1 using AM281, decreases the vasorelaxant effect caused by Capsaicin. On the other hand, the vasorelaxant effect is not altered in the presence of the CB2 receptor antagonist AM630. Furthermore, a partial decrease of the effect of Capsaicin was also seen when L-type calcium channels are blocked. A complete block of Capsaicin-induced vasorelaxation was achieved using a combination of Verapamil and AM281. In accordance to our results, Capsaicin-induced vasorelaxation of the rat aorta is neither dependent of TRPV1 or CB2 receptors, but rather it is strongly suggested that a tandem mechanism between inactivation of L-type calcium channels and the direct activation of CB1 receptors is involved. These findings are supported by CB1 docking simulation which predicted a binding site on CB1 receptors for Capsaicin.

The effect of MD1 on potassium and L-type calcium current of cardiomyocytes from high-fat diet mice

ABSTRACT Myeloid differentiation protein 1 (MD1) is exerted an anti-arrhythmic effect in obese mice. Therefore, we sought to clarify whether MD1 can alter the electrophysiological remodeling of cardiac myocytes from obese mice by regulating voltage-gated potassium current and calcium current. MD1 knock-out (KO) and wild type (WT) mice were given a high-fat diet (HFD) for 20 weeks, starting at the age of 6 weeks. The potential electrophysiological mechanisms were estimated by whole-cell patch-clamp and molecular analysis. After 20-week HFD feeding, action potential duration (APD) from left ventricular myocytes of MD1-KO mice revealed APD20, APD50, and APD90 were profoundly enlarged. Furthermore, HFD mice showed a decrease in the fast transient outward potassium currents (Ito,f), slowly inactivating potassium current (IK, slow), and inward rectifier potassium current (IK1). Besides, HFD-fed mice showed that the current density of ICaL was significantly lower, and the haft inactivation voltage was markedly shifted right. These HFD induced above adverse effects were further exacerbated in KO mice. The mRNA expression of potassium ion channels (Kv4.2, Kv4.3, Kv2.1, Kv1.5, and Kir2.1) and calcium ion channel (Cav1.2) was markedly decreased in MD1-KO HFD-fed mice. MD1 deletion led to down-regulated potassium currents and slowed inactivation of L-type calcium channel in an obese mice model.

Effects of exendin-4 on colonic motility in rats and its underlying mechanism.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists modulate gastrointestinal motility; however, the effects of GLP-1R agonists on colonic motility are still controversial, and the molecular mechanism is unclear. Exendin-4 shares 53% homology with GLP-1 and is a full agonist of GLP-1R. In this study, our aims were to explore the role and mechanism of exendin-4 in isolated rat colonic tissues and cells. An organ bath system was used to examine the spontaneous contractions of smooth muscle strips. The whole-cell patch-clamp technique was used to investigate the currents of L-type voltage-dependent calcium channels and large conductance Ca2+ -activated K+ (BKCa ) channels in smooth muscle cells. Exendin-4 decreased both the amplitude and frequency of spontaneous contractions of smooth muscle strips in a concentration-dependent manner. The inhibitory effect was completely blocked by exendin-4(9-39), a GLP-1R antagonist. Moreover, this effect was partially abolished by tetrodotoxin (TTX), a blocker of neuronal voltage-dependent Na+ channels, Nω-Nitro-l-arginine (L-NNA), a nitric oxide synthase (NOS) inhibitor, apamin, an inhibitor of small-conductance Ca2+ -activated K+ (SK) channels. Whole-cell patch-clamp recordings revealed that exendin-4 inhibited the peak current of L-type calcium channels in colonic smooth muscle cells, but did not change the shape of the current-voltage (I-V) curves. The steady-state activation and steady-state inactivation of L-type calcium channels were not affected. Likewise, BKCa currents were significantly inhibited by exendin-4. Exendin-4 indirectly inhibits colonic muscle activity via a nitrergic and a purinergic neural pathway through NO and ATP release and inhibits L-type voltage-dependent calcium channels and BKCa channels in smooth muscle cells.

Key role of segment IS4 in Cav1.2 inactivation: link between activation and inactivation

Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to −26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of the Boltzmann curve of inactivation was decreased from 17.4 ± 3.5 mV (IS4(K1Q)) to 6.2 ± 0.7 mV (IS4(K1Q/R2Q/R3Q/R4Q)). Neutralizations of single or multiple charges in IIS4 and IIIS4 did not significantly affect the time course of inactivation. Neutralization of individual IVS4 charges shifted the inactivation curve between 17.4 ± 1.7 mV (IVS4(R2Q)) and −4.6 ± 1.4 mV (IVS4(R4Q)) on the voltage axis and affected the slope of the inactivation curves (IVS4(R2Q): 10.2 ± 1.2 mV, IVS4(R4Q): 9.7 ± 0.7 mV and IVS4(K5Q): 8.1 ± 0.7 mV vs WT: 14.1 ± 0.8 mV). IS4(K1Q) attenuated while IS4(K1Q/R2Q/R3Q) and IS4(K1Q/R2Q/R4Q/R3Q) enhanced the development of inactivation. Shifts in the voltage dependence of inactivation curves induced by IS4 neutralizations significantly correlated with shifts of the voltage dependence of channel activation (r = 0.95, p < 0.01) indicating that IS4 movement is not only rate limiting for activation but also initiates inactivation. The paradoxical decrease of the slope factor of the steady-state inactivation and acceleration of inactivation kinetics upon charge neutralization in segment IS4 may reflect the loss of stabilizing interactions of arginines and lysine with surrounding residues.

Structural Characterization of Calmodulin Disease Mutations

Calmodulin (CaM) is a ubiquitous calcium-sensing protein involved in the propagation of intracellular calcium signals and the regulation of events ranging from muscle contraction to cell excitability. The human genome contains three CaM genes (CALM 1-3) which encode for protein with identical primary sequences. Despite the redundancy of CaM, single missense mutations in even one of the six alleles are associated with disease phenotypes such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and early-onset severe long QT syndrome (esLQT). CPVT can lead to stress- and exercise-induced arrhythmias and sudden cardiac death; esLQT is characterized by a prolonged QT interval which can also result in ventricular fibrillation. Despite the devastating genetic disorders associated with CaM mutations, the molecular mechanisms by which these mutations manifest into dominant disease phenotypes have yet to be elucidated. Here, we present the crystal structures of several CaM disease mutants, one of which represents a novel CaM conformation not previously characterized. Significant structural changes are observed in both EF-hands III and IV of the C-lobe. In particular, the mutation disrupts the calcium coordination network in EF-hand III and results in abolished calcium binding, leading to CaM that resembles an intermediate between the Ca2+-CaM and apo-CaM states. In contrast, structures of other disease mutants revealed CaM conformations that closely resemble the wild type structure, with little positional shift in the EF-hand helices of either the N- or C-lobe. These structures can help explain the diverse effects of CaM mutations and the associated disease mechanisms, especially as CaM mutations have differential effects on the function of ryanodine receptor 2 and calcium-dependent inactivation of L-type calcium channels.

Investigation in the Role of Lamin A (LMNA) Deficiency in Heart Block and Dilated Cardiomyopathy (DCM) Using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Investigation in the Role of Lamin A (LMNA) Deficiency in Heart Block and Dilated Cardiomyopathy (DCM) Using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Population Density and Moment-based Approaches to Modeling Domain Calcium-mediated Inactivation of L-type Calcium Channels.

We present a population density and moment-based description of the stochastic dynamics of domain [Formula: see text]-mediated inactivation of L-type [Formula: see text] channels. Our approach accounts for the effect of heterogeneity of local [Formula: see text] signals on whole cell [Formula: see text] currents; however, in contrast with prior work, e.g., Sherman et al. (Biophys J 58(4):985-995, 1990), we do not assume that [Formula: see text] domain formation and collapse are fast compared to channel gating. We demonstrate the population density and moment-based modeling approaches using a 12-state Markov chain model of an L-type [Formula: see text] channel introduced by Greenstein and Winslow (Biophys J 83(6):2918-2945, 2002). Simulated whole cell voltage clamp responses yield an inactivation function for the whole cell [Formula: see text] current that agrees with the traditional approach when domain dynamics are fast. We analyze the voltage-dependence of [Formula: see text] inactivation that may occur via slow heterogeneous domain [[Formula: see text]]. Next, we find that when channel permeability is held constant, [Formula: see text]-mediated inactivation of L-type channels increases as the domain time constant increases, because a slow domain collapse rate leads to increased mean domain [[Formula: see text]] near open channels; conversely, when the maximum domain [[Formula: see text]] is held constant, inactivation decreases as the domain time constant increases. Comparison of simulation results using population densities and moment equations confirms the computational efficiency of the moment-based approach, and enables the validation of two distinct methods of truncating and closing the open system of moment equations. In general, a slow domain time constant requires higher order moment truncation for agreement between moment-based and population density simulations.

Hydrogen sulfide regulates the colonic motility by inhibiting both L-type calcium channels and BKCa channels in smooth muscle cells of rat colon.

ObjectiveTo examine the hypothesis that hydrogen sulfide (H2S) regulates the colonic motility by modulating both L-type voltage-dependent calcium channels and large conductance Ca2+-activated K+ (BKCa) channels.MethodsImmunohistochemistry was performed on rat colonic samples to investigate the localization of the H2S-producing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). The contractions of proximal colonic smooth muscle were studied in an organ bath system. The whole-cell patch-clamp technique was used to record both L-type calcium currents (I Ca,L) and BKCa currents in colonic smooth muscle cells (SMCs) isolated from male Wistar rats.ResultsImmunohistochemistry revealed the presence of CBS and CSE in mucosa, smooth muscle cells and myenteric neurons. The H2S donor NaHS inhibited spontaneous contractions of the longitudinal muscle and circular muscle strips in a dose-dependent manner, and the inhibitory effects were not blocked by tetrodotoxin. NaHS inhibited the peak I Ca,L in colonic SMCs at a membrane potential of 0 mV. The current-voltage (I-V) relationship of L-type calcium channels was modified by NaHS, and the peak of the I-V curve was shifted to the right. NaHS (200μΜ) evoked a significant rightward shift of the steady-state activation curve and inhibited the inactivation of L-type calcium channels. Furthermore, NaHS reversibly decreased the peak I Ca,L in a dose-dependent manner. Likewise, BKCa channels were significantly inhibited by NaHS, and the addition of NaHS caused a time- and dose-dependent reduction in the BKCa current.ConclusionThe relaxant effect of H2S on colonic muscle strips may be associated with the direct inhibition of H2S on L-type calcium channels. H2S may be involved in the regulation of calcium homeostasis in colonic SMCs of rat colon.

A Population Density and Moment-Based Approach to Modeling Domain Calcium-Mediated Inactivation of L-Type Calcium Channels

We present a population density and moment-based description of stochastic domain calcium-mediated inactivation of L-type calcium channels. Our approach accounts for the effect of heterogeneity of local calcium signals on whole cell calcium currents; however, in contrast with prior work by Sherman et al. [Biophys J. 58(4):985, 1990], we do not assume that calcium domain formation and collapse are fast compared to channel gating. We demonstrate the population density and moment-based modeling approach using a12-state Markov chain model of an L-type calcium channel [Greenstein and Winslow, Biophys J. 83(6):2918, 2002]. Simulated whole cell voltage clamp responses yield an inactivation function for the whole cell calcium current that agrees or disagrees with the classic result of Sherman et al. when domains dynamics are fast or slow, respectively. We analyze the voltage-dependence of calcium inactivation that occurs via slow heterogeneous domains and find that when channel permeability is held constant, calcium inactivation increases as the domain time constant increases. However, when this parameter study is repeated for fixed maximum domain calcium concentration, inactivation decreases as the domain time constant increases. Comparison of simulation results using population densities and moment equations confirms the computational efficiency of the moment-based approach, and enables the validation of several distinct methods of truncating and closing the open system of moment equations. In general, a slow domain time constant requires higher order moment truncation for agreement between moment-based and population density simulations.

The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

Cholestrol Depletion reduces Voltage-Dependent Inactivation of L-Type Calcium Channel and DHEA Augments the Inacivation in A7R5 Cells

Cholesterol (Chol), a structural component of the membrane, is involved also in the functional regulation of ion channels. The increase of membrane Chol is associated with the reduction of L-type Ca2+ channel current (ICa,L). The effect of Chol on the voltage-dependent inactivation is not known, however. Dehydroepiandrosterone (DHEA), a cholesterol-derived adrenal steroid hormone, produces a voltage-dependent inhibition of ICa,L at micro-molar concentrations. We studied the effect of Chol on the inactivation of ICa,L and on the DHEA-induced inhibition of ICa,L in A7r5, a cell line derived from fetal rat aorta. Chol was depleted with methyl-beta-cyclodextrin (CD) or loaded by cholesterol bound CD (chol-CD). Whole cell ICa,L was recorded with 10 mM Ba2+ as the charge carriers. T-type Ca2+ channel current was inactivated by 50 ms pre-pulse to −40 mV. The maximal density of ICa,L was decreased by 55% by loading Chol with 30 mM chol-CD, while it was not affected by the depletion of Chol by 10 and 30 mM CD. The depletion of Chol by 30 mM CD remarkably decreased the rate of inactivation of ICa,L, generating sustained current. It shifted V0.5 in the f∞-V curve to a depolarizing direction by 10 mV from control −30 mV, with appearance of 27% non-inactivating component at 0 mV. DHEA (30 micro M) inhibited the maximal amplitude of ICa,L by ∼30% irrespective of the manipulation of Chol. It shifted the V0.5 to a hyperpolarizing direction by 6 mV in control but 10 mV in the 30 mM CD treated cells. We conclude that membrane cholesterol is essential for the normal voltage-dependent inactivation of L-type Ca2+ channel. DHEA, at pharmacological concentrations, more potently augments the inactivation in the cholesterol depleted cells.

Inactivation of L-type calcium channel modulated by HCN2 channel

Ca(2+) entry is delicately controlled by inactivation of L-type calcium channel (LTCC) composed of the pore-forming subunit alpha1C and the auxiliary subunits beta1 and alpha2delta. Calmodulin is the key protein that interacts with the COOH-terminal motifs of alpha1C, leading to the fine control of LTCC inactivation. In this study we show evidence that a hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, can act as a nonchannel regulatory protein to narrow the L-type Ca(2+) channel current-voltage curve. In the absence of LTCC auxiliary subunits, HCN2 can induce alpha1C inactivation. Without alpha2delta, HCN2-induced fast inactivation of alpha1C requires calmodulin. With alpha2delta, the alpha1C/HCN2/alpha2delta channel inactivation does not require calmodulin. In contrast, beta1-subunit plays a relatively minor role in the interaction of alpha1C with HCN2. The NH(2) terminus of HCN2 and the IQ motif of alpha1C subunit are required for alpha1C/HCN2 channel interaction. Ca(2+) channel inactivation is significantly slowed in hippocampus neurons (HNs) overexpressing HCN2 mutant lacking NH(2) terminus and accelerated in HNs overexpressing the wild-type HCN2 compared with HN controls. Collectively, these results revealed a potentially novel protection mechanism for achieving the LTCC inactivation via interaction with HCN2.

Gamma1 Subunit Renders Cav1.2 Channels Dependent on Cell Cycle

Auxiliary γ subunits are known to enhance inactivation of L-type calcium channels. When co-expressed in tsA-201 cells with α1C and β2a subunits, the γ1 subunit shifts the voltage-dependence of inactivation by about −20 mV. The voltage of half-maximal inactivation, V1/2, was determined with 5 s long conditioning pre-pulses. On average, addition of γ1 to α1C/β2a channels changed V1/2 from −24±5 mV (n=30) to −44±12 mV (n=92). We noticed that V1/2, but not the steepness of the voltage-dependence of inactivation, varies greatly in cells with γ1 and set up to find the cause of the cell-to-cell variability. Serum starvation and “ER shock” by the N-glycosylation blocker tunicamicin further shifted inactivation to negative voltages. The average V1/2 was −59±12 mV (n=12) in serum-free and −69±13 mV(n=32) in tunicamicin treated cells. These treatments altered inactivation only when γ1 was present and the effects were similar when β3 substituted for β2a. Mutations of γ1 that remove consensus N-glycosylation sites had only partial effect (V1/2=-60±18 mV, n=29) and did not reduce the cell-to-cell variability, indicating that N-glycosylation of γ1 was not its primary cause.Serum starvation and tunicamicin are known to produce cell-cycle arrest in the G0/G1 phase and, therefore, could act on γ1 indirectly by interfering with a cell-cycle dependent pathway. We characterized inactivation in cells expressing fluorescent probes visualizing cell-cycle activity. In support of our hypothesis, V1/2 was −55±16 mV, n=32, in G1 and −36±7 mV, n=20, in S/G2/M cells.Therefore, we propose that a novel cell-cycle dependent regulatory pathway controls voltage-dependent inactivation and functional availability of L-type calcium channels in the presence of γ1 subunit.The project was supported by MH079406 from NIMH.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant β1 subunits

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.