L-type Calcium Channel Gating Research Articles

Electrophysiological effects of stretch-activated ion channels: a systematic computational characterization.

Cardiac electrophysiology and mechanics are strongly interconnected. Their interaction is, among others, mediated by mechano-electric feedback through stretch-activated ion channels (SACs). The electrophysiological changes induced by SACs may contribute to arrhythmogenesis, but the precise SAC-induced electrophysiological changes remain incompletely understood. Here, we provide a systematic characterization of stretch effects through three distinguished SACs on cardiac electrophysiology using computational modelling. We implemented potassium-selective, calcium-selective and non-selective SACs in the Tomek-Rodriguez-O'Hara-Rudy model of human ventricular electrophysiology. The model was calibrated to experimental data from isolated cardiomyocytes undergoing stretch, considering inter-species differences, and disease-related remodelling of SACs. SAC-mediated effects on the action potential (AP) were analysed by varying stretch amplitude, application timing and/or duration. Afterdepolarizations of different amplitudes were observed with transient 10-ms stretch stimuli of 15-18% applied during phase 4, while stretch ≥18% during phase 4 elicited triggered APs. Longer stimuli shifted the threshold of AP trigger during phase 4 to lower amplitudes, while shorter stimuli increased it. Continuous stretch provoked electrophysiological remodelling. Furthermore, stretch shortened duration or changed morphology of a subsequent electrically evoked AP, and, if applied during a vulnerable time window with sufficient amplitude, prevented its occurrence because of stretch-induced modulation of sodium and L-type calcium channel gating. These effects were more pronounced with disease-related SAC remodelling due to increased stretch sensitivity of diseased hearts. We showed that SACs may induce afterdepolarizations and triggered activities, and prevent subsequent AP generation or change its morphology. These effects depend on cardiomyocyte stretch characteristics and disease-related SACs remodelling and may contribute to cardiac arrhythmogenesis. KEY POINTS: The interplay between cardiac electrophysiology and mechanics is mediated by mechano-electric feedback through stretch-activated ion channels (SACs). These channels may be pro-arrhythmic, but their precise effect on electrophysiology remains unclear. Here we present a systematic in silico characterization of stretch effects through three SACs by implementing inter-species differences as well as disease-related remodelling of SACs in a novel computational model of human ventricular cardiomyocyte electrophysiology. Our simulations showed that, at the cellular level, SACs may provoke electrophysiological remodelling, afterdepolarizations, triggered activities, change the morphology or shorten subsequent electrically evoked action potentials. The model further suggests that a vulnerable window exists in which stretch prevents the following electrically triggered beat occurrence. The pro-arrhythmic effects of stretch strongly depend on disease-related SAC remodelling as well as on stretch characteristics, such as amplitude, time of application and duration. Our study helps in understanding the role of stretch in cardiac arrhythmogenesis and revealing the underlying cellular mechanisms.

L-Type Calcium Channel Gating Modifiers as a New Class of Antiarrhythmic Drugs

Pharmacological treatment of cardiac arrhythmias includes the use of Ca2+ channel blockers (Class IV antiarrhythmics), that while suppressing arrhythmias, also block peak ICa,L, compromising EC coupling and contractility. To circumvent these limitations, we are studying and testing the properties of L-type Ca channel gating modifiers, rather than blockers, to selectively reduce the non-inactivating component (late ICa,L), leaving peak ICa,L minimally affected, thus preserving cardiac contractility. This strategy is based on our discovery that the selective reduction of late ICa,L potently suppress early afterdepolarizations (EADs) of the cardiac action potential (AP), recognized cellular triggers of arrhythmias (Madhvani et al., 2015). We previously reported that roscovitine, a purine analog known to reduce late ICa,L, potently suppressed H2O2-mediated EADs in rabbit ventricular myocytes. We are evaluating the robustness of this pharmacological intervention in a new model of EAD favoring condition using 600μM H2O2+1-2μM Isoproterenol. H2O2 alone (n=7) caused EADs in 83.2±5.39% APs and 85.7±7.83% APs in presence of H2O2+Isoproterenol (n=5); the perfusion of roscovitine (20 µM) efficiently suppressed EAD occurrence restoring normal AP duration in all myocytes tested. This antiarrhythmic effect was achieved without significantly perturbing calcium transients and cell shortening, indicating maintenance of normal EC coupling. We also tested roscovitine ability to suppress EAD-triggered ventricular fibrillation (VF) in isolated-perfused aged rat and rabbit hearts. We found that roscovitine suppressed VF induced by H2O2 (8/8 rat hearts, average time: 13±2.8 min) and H2O2+Isoproterenol (4/4 rabbit hearts, average time: 21±7 min), restoring sinus rhythm. Roscovitine had no significant effect on the rate-pressure product (RPP) as a measure of myocardial workload in isolated rat hearts (n=5). We propose that these findings establish roscovitine as a pilot compound to drive the development of a new class of antiarrhythmics based on Ca channel gating modification.

Peptidomimetic Targeting of Cavβ2 Overcomes Dysregulation of the L-Type Calcium Channel Density and Recovers Cardiac Function.

L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavβ2 chaperone regulates channel density at the plasma membrane. On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavβ2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavβ2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavβ2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavβ2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavβ2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavβ2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.

Structural determinants of CaV1.3 L-type calcium channel gating

A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Cav1.3 L-type Ca2+ channels, in particular the voltage dependence of activation (V0.5) and Ca2+ dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Cav1.3 (Cav1.3L), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCav1.3scg). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCav1.3scg with a rat cDNA isolated from rat pancreas (rCav1.3L). When expressed in tsA-201 cells under identical experimental conditions rCav1.3L exhibited Ca2+ current properties indistinguishable from human and mouse Cav1.3L, compatible with the presence of a functional CTM. In contrast, rCav1.3scg showed gating properties similar to human short splice variants lacking a CTM. rCav1.3scg differs from rCav1.3L at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCav1.3scg explained most of the functional differences to rCav1.3L. Their mutation to the corresponding residues in rCav1.3L (G244, V2075) revealed that both contributed to the more negative V0.5, but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCav1.3L. We found no evidence for transcripts containing one of the single rCav1.3scg mutations in rat superior cervical ganglion preparations. However, the rCav1.3scg variant provided interesting insight into the structural machinery involved in Cav1.3 gating.

Structural determinants of CaV1.3 L-type calcium channel gating

Cav1.3 L-type Ca2+-channels are key signal molecules for hearing, cardiac pacemaking and neuronal excitability. We have recently discovered a C-terminal intramolecular protein-interaction in Cav1.3 α1-subunits as a gating modifier (CTM). This modulatory domain is absent in short C-terminal splice variants. Its presence shifts half maximal activation voltage (V0.5) to more positive potentials and inhibits Ca2+-dependent-inactivation (CDI). This regulation is present in long variants of human (Singh et al., 2008) and rat cDNA clones from pancreatic islets (rCav1.3pan) but not in a clone derived from superior cervical ganglion (rCav1.3scg). rCav1.3scg differs from rCav1.3pan at three amino acid (aa) positions (S244G, V1104A, A2073V), a polymethionine-stretch with two additional lysines, and one alternatively spliced locus (exon 31). We took advantage of these discrete differences to determine their role for the kinetic differences between rCav1.3scg and rCav1.3pan (in voltage-dependence of activation and CDI) by expressing channel mutants in tsA-201 cells and whole-cell patch-clamp-recordings.Two aa (S244, A2073) in rCav1.3scg explained most of the functional differences to rCav1.3pan. Mutation S244G even further enhanced CDI of rCav1.3scg and shifted its V0.5 to more positive potentials. A2073V (located within the CTM) also shifted V0.5 more positive but almost eliminated CDI. The cooperative action in the double-mutant restored gating properties (CDI, V0.5) similar to rCav1.3pan. Their effects are compatible with a recently proposed allosteric CDI mechanism implying CaM-mediated inhibition of the activation gate (Tadross and Yue, 2010). Analysis of inactivation kinetics suggest that G244 decreases the open probability (Po) of the inactivated gating mode, whereas A2073V reduces the maximal Po of rCav1.3scg as evident from increased gating currents.G244 and V2073 affect CDI through different molecular mechanisms. Together they stabilize the gating behaviour of long CaV1.3 splice variants undergoing C-terminal-modulation.Support: Austrian Science Fund (F4402, P20670, W11), University of Innsbruck

L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells

Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G(q/11). In the present study we show that the Ca(2+) current flowing through L-type voltage-gated Ca(2+) channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca(2+) channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.

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.

Protein kinase G reverses all isoproterenol induced changes of cardiac single L-type calcium channel gating.

cGMP reduces the effect of beta-adrenoceptor agonists on cardiac L-type calcium current by protein kinase G activation. Stimulation of beta-adrenoceptors increases protein kinase A dependent phosphorylation of L-type calcium channels via cAMP. At the single channel level, protein kinase A dependent phosphorylation increases both availability and open probability. The present study investigates how cGMP antagonises protein kinase A induced changes of single L-type calcium channel gating. Single L-type calcium channels were recorded in the cell attached configuration of the patch clamp technique in isolated mouse ventricular myocytes. The beta-adrenoceptor agonist isoproterenol (10(-6) M) enhanced single channel peak average current by increasing availability and open probability and decreasing the time constant of long close times. 8-Br-cGMP (10(-3) M) completely reversed these effects. The phosphatase inhibitor okadaic acid (10(-6) M) did not influence the effect of 8-Br-cGMP. The protein kinase G inhibitor Rp-8Br-PET-cGMPS (10(-7) M) abated the effect of 8-Br-cGMP. Activation of protein kinase A by the hydrolysis-resistant cAMP derivative 8-Br-cAMP (10(-3) M) enhanced L-type calcium channel activity like isoproterenol and its effect was also reversed by 8-Br-cGMP. 8-Br cGMP diminishes beta-adrenoceptor activation of L-type calcium channels via protein kinase G. It interacts with the beta-adrenoceptor signaling pathway distal of adenylyl cyclase. Our observations suggest that protein kinase G interacts either with protein kinase A or directly with the L-type calcium channel.

Properties of Ca2+ sparks evoked by action potentials in mouse ventricular myocytes.

1. Calcium sparks were examined in enzymatically dissociated mouse cardiac ventricular cells using the calcium indicator fluo-3 and confocal microscopy. The properties of the mouse cardiac calcium spark are generally similar to those reported for other species. 2. Examination of the temporal relationship between the action potential and the time course of calcium spark production showed that calcium sparks are more likely to occur during the initial repolarization phase of the action potential. The latency of their occurrence varied by less than 1.4 ms (s.d.) and this low variability may be explained by the interaction of the gating of L-type calcium channels with the changes in driving force for calcium entry during the action potential. 3. When fixed sites within the cell are examined, calcium sparks have relatively constant amplitude but the amplitude of the sparks was variable among sites. The low variability of the amplitude of the calcium sparks suggests that more than one sarcoplasmic reticulum (SR) release channel must be involved in their genesis. Noise analysis (with the assumption of independent gating) suggests that > 18 SR calcium release channels may be involved in the generation of the calcium spark. At a fixed site, the response is close to 'all-or-none' behaviour which suggests that calcium sparks are indeed elementary events underlying cardiac excitation-contraction coupling. 4. A method for selecting spark sites for signal averaging is presented which allows the time course of the spark to be examined with high temporal and spatial resolution. Using this method we show the development of the calcium spark at high signal-to-noise levels.

Critical role of conserved proline residues in the transmembrane segment 4 voltage sensor function and in the gating of L-type calcium channels.

The fourth transmembrane segment (S4) has been shown to function as a voltage sensor in voltage-gated channels. On membrane depolarization, a stretch of S4 moves outward and initiates a number of conformational changes that ultimately lead to channel opening. Conserved proline residues are in the middle of the S4 of motifs I and III in voltage-dependent Ca2+ channels. Because proline often introduces a "kink" into a helical structure of proteins, these residues might have an intrinsic function in the voltage sensor. Here, we report that the removal of S4 prolines results in a dramatic shortening of channel open time whereas the introduction of extra prolines to the corresponding positions in motif IIS4 and IVS4 lengthens channel open time. The number of S4s with a proline residue showed a clear positive correlation with the mean open time of the channel. The mean open time was >11-fold longer for a channel mutagenized to have prolines in all four S4s compared with a channel that had no prolines in the S4 region. Additionally, prolines in the S4s slowed activation kinetics and shifted the voltage dependence of activation and inactivation in a hyperpolarized direction. Our results strongly suggest that proline residues in the S4s are critical for stabilizing the open state of the channel. Moreover, it is suggested that motif IS4 and IIIS4 contribute to the channel opening more efficiently than motif IIS4 and IVS4.

Numerical simulation of local calcium movements during L-type calcium channel gating in the cardiac diad

Computer simulation was used to investigate the calcium levels after sarcolemmal calcium influx through L-type calcium channels (DHPRs) into the narrow diadic space of cardiac muscle. The effect of various cytosolic and membranebound buffers, diad geometry, DHPR properties (open time and current), and surface charge were examined. The simulations showed that phospholipid binding sites on the sarcolemmal membrane are the major buffer affecting free calcium ([Ca2+]) levels in the diad. The inclusion of surface charge effects calculated from Gouy-Chapman theory resulted in a marked decrease in [Ca2+] levels at all times and a faster decay of [Ca2+] after termination of DHPR influx. For a DHPR current of 200 fA, [Ca2+] at the center of the diad reached peak levels of approximately 73 microM. In larger diads (> or = 400 nm diameter), [Ca2+] decayed more slowly than in smaller diads (100-200 nm diameter), although peak [Ca2+] levels reached during typical DHPR open times were similar. For a wide range of DHPR single-channel current magnitudes (Ica = 25-200 fA), [Ca2+] levels in the diad were approximately proportional to ICa. The decrease in calculated [Ca2+] levels due to the effects of surface charge can be interpreted as resulting from an effective "volume expansion" of the diad space. Furthermore, the layer of increased [Ca2+] close to the sarcolemmal membrane can act as a fast buffer.

Isolation of myocardial L-type calcium channel gating currents with the spider toxin omega-Aga-IIIA.

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents. omega-Aga-IIIA permits the study of L-type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, omega-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Q(on)) and after repolarization (Q(off)). Q(on) and Q(off) are suppressed by 1 microM felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Q(on) is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Q(off) decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Q(on) and Q(off) indicate that they reflect gating currents associated with L-type Ca channels. omega-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using omega-Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that L- and T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account.