Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.     

The creatine kinase system is essential for optimal refill of the sarcoplasmic reticulum Ca2+ store in skeletal muscle.

Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca(2+) cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca(2+) homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca(2+) transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca(2+) probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca(2+) release channels, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca(2+) load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca(2+) removal rate and the amount of Ca(2+) released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca(2+) signals.     

RyRCa2+ leak limits cardiac Ca2+ window current overcoming the tonic effect of calmodulinin mice

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.     

Determinants for calmodulin binding on voltage-dependent Ca2+ channels

Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.     

Calcium flux in turtle ventricular myocytes

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.     

A helix-breaking mutation in the epithelial Ca(2+) channel TRPV5 leads to reduced Ca(2+)-dependent inactivation

TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation.     

Contribution of the Na+/Ca2+ exchanger to rapid Ca2+ release in cardiomyocytes

Trigger Ca(2+) is considered to be the Ca(2+) current through the L-type Ca(2+) channel (LTCC) that causes release of Ca(2+) from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current (I(Ca)). In this article, we investigate the contribution of the Na(+)/Ca(2+) exchanger (NCX) to the trigger Ca(2+). Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na(+)/Ca(2+) exchange delays the Ca(2+) transient by 3-4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na(+) and Ca(2+) is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na(+) channel is present in the fuzzy space, the NCX is able to bring enough Ca(2+) into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na(+) channel and NCX.     

Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.     

Glu¹⁰⁶ in the Orai1 pore contributes to fast Ca²⁺-dependent inactivation and pH dependence of Ca²⁺ release-activated Ca²⁺ (CRAC) current

FCDI (fast Ca2?-dependent inactivation) is a mechanism that limits Ca2? entry through Ca2? channels, including CRAC (Ca2? release-activated Ca2?) channels. This phenomenon occurs when the Ca2? concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca2? sensor that communicates the Ca2? load of the intracellular stores to Orai1, have been shown to regulate fast Ca2?-dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca2?-dependent inactivation in this channel are not well understood. We have identified that a pore mutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca2?-binding site that regulates fast Ca2?-dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca2?-dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC. Unlike WT ICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na? permeation through the E106D Orai1 pore by Ca2? is diminished. These results suggest that Glu1?? inside the CRAC channel pore is involved in co-ordinating the Ca2?-binding site that mediates fast Ca2?-dependent inactivation.     

Mitochondrial regulation of store-operated CRAC channels

In eukaryotic cells, one major route for Ca(2+) influx is through store-operated CRAC channels, which are activated following a fall in Ca(2+) content within the endoplasmic reticulum. Mitochondria are key regulators of this ubiquitous Ca(2+) influx pathway. Respiring mitochondria rapidly take up some of the Ca(2+) released from the stores, resulting in more extensive store depletion and thus robust activation of CRAC channels. As CRAC channels open, the ensuing rise in cytoplasmic Ca(2+) feeds back to inactivate the channels. By buffering some of the incoming Ca(2+) mitochondria reduce Ca(2+)-dependent inactivation of the CRAC channels, resulting in more prolonged Ca(2+) influx. However, mitochondria can release Ca(2+) close to the endoplasmic reticulum, accelerating store refilling and thus promoting deactivation of the CRAC channels. Mitochondria thus regulate all major transitions in CRAC channel gating, revealing remarkable versatility in how this organelle impacts upon Ca(2+) influx. Recent evidence suggests that mitochondria also control CRAC channels through mechanisms that are independent of their Ca(2+)-buffering actions and ability to generate ATP. Furthermore, pyruvic acid, a key intermediary metabolite and precursor substrate for the Krebs cycle, reduces the extent of Ca(2+)-dependent inactivation of CRAC channels. Hence mitochondrial metabolism impacts upon Ca(2+) influx through CRAC channels and thus on a range of key downstream cellular responses.     

Role of the Na(+)-Ca(2+) exchanger as an alternative trigger of CICR in mammalian cardiac myocytes

Ca(2+) influx through the L-type Ca(2+) channels is the primary pathway for triggering the Ca(2+) release from the sarcoplasmic reticulum (SR). However, several observations have shown that Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger current (I(Na-Ca)) could also trigger the Ca(2+) release. The aim of the present study was to quantitate the role of this alternative pathway of Ca(2+) influx using a mathematical model. In our model 20% of the fast sodium channels and the Na(+)-Ca(2+) exchanger molecules are located in the restricted subspace between the sarcolemma and the SR where triggering of the calcium-induced calcium release (CICR) takes place. After determining the strengths of the alternative triggers with simulated voltage-clamps in varied membrane voltages and resting [Na](i) values, we studied the CICR in simulated action potentials, where fast sodium channel current contributes [Na](i) of the subspace. In low initial [Na](i) the Ca(2+) influx via the L-type Ca(2+) channels is the major trigger for Ca(2+) release from the SR, and the Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger cannot trigger the CICR. However, depending on the initial [Na](i), the contribution of the Ca(2+) entry via the exchanger may account for 25% (at [Na](i) = 10 mM) to nearly 100% ([Na](i) = 30 mM) of the trigger Ca(2+). The shift of the main trigger from L-type calcium channels to the exchanger reduced the delay between the action potential upstroke and the intracellular calcium transient. This may contribute to the function of the myocyte in physiological situations where [Na](i) is elevated. These main results remain the same when using different estimates for the most crucial parameters in the modeling or different models for the exchanger.     

Capacitative calcium entry in the nervous system

Capacitative calcium entry is a process whereby the depletion of Ca(2+) from intracellular stores (likely endoplasmic or sarcoplasmic reticulum) activates plasma membrane Ca(2+) channels. Current research has focused on identification of capacitative calcium entry channels and the mechanism by which Ca(2+) store depletion activates the channels. Leading candidates for the channels are members of the transient receptor potential (TRP) superfamily, although no single gene or gene product has been definitively proven to mediate capacitative calcium entry. The mechanism for activation of the channels is not known; proposals fall into two general categories, either a diffusible signal released from the Ca(2+) stores when their Ca(2+) levels become depleted, or a more direct protein-protein interaction between constituents of the endoplasmic reticulum and the plasma membrane channels. Capacitative calcium entry is a major mechanism for regulated Ca(2+) influx in non-excitable cells, but recent research has indicated that this pathway plays an important role in the function of neuronal cells, and may be important in a number of neuropathological conditions. This review will summarize some of these more recent findings regarding the role of capacitative calcium entry in normal and pathological processes in the nervous system.     

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.     

Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles

Fast Ca(2+) release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca(2+). Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca(2+)] and [Mg(2+)]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa < or =5. These results suggest that channels respond differently when exposed to sudden [Ca(2+)] changes than when exposed to Ca(2+) for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg(2+) produced a marked inhibition of release kinetics at pCa 6 (K(0.5) = 63 microM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg(2+)] tested and increased at pCa 6 the K(0.5) for Mg(2+) inhibition, from 63 microM to 136 microM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca(2+) and Mg(2+) followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.     

Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity

Obesity has been shown to impair myocardial performance. Nevertheless, the mechanisms underlying the participation of calcium (Ca(2+) ) handling on cardiac dysfunction in obesity models remain unknown. L-type Ca(2+) channels and sarcoplasmic reticulum (SR) Ca(2+) -ATPase (SERCA2a), may contribute to the cardiac dysfunction induced by obesity. The purpose of this study was to investigate whether myocardial dysfunction in obese rats is related to decreased activity and/or expression of L-type Ca(2+) channels and SERCA2a. Male 30-day-old Wistar rats were fed standard (C) and alternately four palatable high-fat diets (Ob) for 15 weeks. Obesity was determined by adiposity index and comorbidities were evaluated. Myocardial function was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic and lusitropic maneuvers. L-type Ca(2+) channels and SERCA2a activity were determined using specific blockers, while changes in the amount of channels were evaluated by Western blot analysis. Phospholamban (PLB) protein expression and the SERCA2a/PLB ratio were also determined. Compared with C rats, the Ob rats had increased body fat, adiposity index and several comorbidities. The Ob muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus and increased extracellular Ca(2+) was compromised. The diltiazem promoted higher inhibition on developed tension in obese rats. In addition, there were no changes in the L-type Ca(2+) channel protein content and SERCA2a behavior (activity and expression). In conclusion, the myocardial dysfunction caused by obesity is related to L-type Ca(2+) channel activity impairment without significant changes in SERCA2a expression and function as well as L-type Ca(2+) protein levels.     

Endogenous cytosolic Ca(2+) buffering is necessary for TRPM4 activity in cerebral artery smooth muscle cells

The melastatin transient receptor potential (TRP) channel, TRPM4, is a critical regulator of smooth muscle membrane potential and arterial tone. Activation of the channel is Ca(2+)-dependent, but prolonged exposures to high global Ca(2+) causes rapid inactivation under conventional whole-cell patch clamp conditions. Using amphotericin B perforated whole cell patch clamp electrophysiology, which minimally disrupts cytosolic Ca(2+) dynamics, we recently showed that Ca(2+) released from 1,2,5-triphosphate receptors (IP(3)R) on the sarcoplasmic reticulum (SR) activates TRPM4 channels, producing sustained transient inward cation currents (TICCs). Thus, Ca(2+)-dependent inactivation of TRPM4 may not be inherent to the channel itself but rather is a result of the recording conditions. We hypothesized that under conventional whole-cell configurations, loss of intrinsic cytosolic Ca(2+) buffering following cell dialysis contributes to inactivation of TRPM4 channels. With the inclusion of the Ca(2+) buffers ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 10mM) or bis-ethane-N,N,N',N'-tetraacetic acid (BAPTA, 0.1mM) in the pipette solution, we mimic endogenous Ca(2+) buffering and record novel, sustained whole-cell TICC activity from freshly-isolated cerebral artery myocytes. Biophysical properties of TICCs recorded under perforated and whole-cell patch clamp were nearly identical. Furthermore, whole-cell TICC activity was reduced by the selective TRPM4 inhibitor, 9-phenanthrol, and by siRNA-mediated knockdown of TRPM4. When a higher concentration (10mM) of BAPTA was included in the pipette solution, TICC activity was disrupted, suggesting that TRPM4 channels on the plasma membrane and IP(3)R on the SR are closely opposed but not physically coupled, and that endogenous Ca(2+) buffer proteins play a critical role in maintaining TRPM4 channel activity in native cerebral artery smooth muscle cells.     

Voltage- and calcium-dependent inactivation in high voltage-gated Ca(2+) channels

Calcium influx into cardiac myocytes via voltage-gated Ca channels is a key step in initiating the contractile response. During prolonged depolarizations, toxic Ca(2+) overload is prevented by channel inactivation occurring through two different processes identified by their primary trigger: voltage or intracellular Ca(2+). In physiological situations, cardiac L-type (Ca(V)1.2) Ca(2+) channels inactivate primarily via Ca(2+)-dependent inactivation (CDI), while neuronal P/Q (Ca(V)2.1) Ca(2+) channels use preferentially voltage-dependent inactivation (VDI). In certain situations however, these two types of channels have been shown to be able to inactivate by both processes. From a structural view point, the rearrangement occurring during CDI and VDI is not precisely known, but functional studies have underlined the role played by at least 2 channel sequences: a C-terminal binding site for the Ca(2+) sensor calmodulin, essential for CDI, and the loop connecting domains I and II, essential for VDI. The conserved regulation of VDI and CDI by the auxiliary channel beta subunit strongly suggests that these two mechanisms may use a set of common protein-protein interactions that are influenced by the auxiliary subunit. We will review our current knowledge of these interactions. New data are presented on L-P/Q (Ca(V)1.2/Ca(V)2.1) channel chimera that confirm the role of the I-II loop in VDI and CDI, and reveal some of the essential steps in Ca(2+) channel inactivation.     

Calcium-transporting systems and calcium regulation in cardiomyocytes

The review systematizes and analyzes recent data about the role of different Ca(2+)-transport mechanisms in the regulation of Ca2+ metabolism and functional activity of the cardiomyocytes. During the cardiac action potential, Ca2+ enters the cardiomyocytes through sarcolemmal L-type calcium channels and via the Na+/Ca2+ exchange. This Ca2+ activates the release of additional Ca2+ from the sarcoplasmic reticulum. The sum of calcium from sarcolemmal influxes and sarcoplasmic release produces contractile effect. For the occurrence of relaxation, Ca2+ remove from the cytoplasm by three mechanisms, namely, sarcoplasmic Ca2+ pump, Na+/Ca2+ exchange and sarcolemmal Ca2+ pump. In this review, the structural and functional properties of the Ca2+ transport systems in the sarcolemmal membranes, sarcoplasmic reticulum and mitochondria are discussed. In addition alterations in regulation of intracellular calcium by activation of beta- and alpha-adrenergic receptors are consider.