Cooperation of two-domain Ca(2+) channel fragments in triad targeting and restoration of excitation- contraction coupling in skeletal muscle

The specific incorporation of the skeletal muscle voltage-dependent Ca(2+) channel in the triad is a prerequisite of normal excitation-contraction (EC) coupling. Sequences involved in membrane expression and in targeting of Ca(2+) channels into skeletal muscle triads have been described in different regions of the alpha(1S) subunit. Here we studied the targeting properties of two-domain alpha(1S) fragments, green fluorescent protein (GFP)-I x II (1-670) and III x IV (691-1873) expressed alone or in combination in dysgenic (alpha(1S)-null) myotubes. Immunofluorescence analysis showed that GFP-I x II or III x IV expressed separately were not targeted into triads. In contrast, on coexpression the two alpha(1S) fragments were colocalized with one another and with the ryanodine receptor in the triads. Coexpression of GFP-I x II and III x IV also fully restored Ca(2+) currents and depolarization-induced Ca(2+) transients, despite the severed connection between the two channel halves and the absence of amino acids 671-690 from either alpha(1S) fragment. Thus, triad targeting, like the rescue of function, requires the cooperation and coassembly of the two complementary channel fragments. Transferring the C terminus of alpha(1S) to the N-terminal two-domain fragment (GFP-I x II x tail), or transferring the I-II connecting loop containing the beta interaction domain to the C-terminal fragment (III x IV x beta in) did not improve the targeting properties of the individually expressed two-domain channel fragments. Thus, the cooperation of GFP-I.II and III.IV in targeting cannot be explained solely by a sequential action of the beta subunit by means of the I-II loop in releasing the channel from the sarcoplasmic reticulum and of the C terminus in triad targeting.     

Trans-sarcolemmal Ca2+ movements associated with contraction of the rabbit right ventricular wall

The purpose of this study was to examine the movements of Ca2+ into the myocardium from the extracellular space during the course of muscle contraction. Equilibration of the rabbit right ventricular wall with perfusate containing 45Ca was measured by collecting effluent droplets over time. This protocol was carried out in a quiescent muscle and then repeated 15-20 minutes later in an identical fashion except that halfway through the collection period the muscle was stimulated to contract. We were thus able to quantitate the contraction-dependent changes in 45Ca movements. In control experiments using [58Co]EDTA as an extracellular space marker, we observed that contraction altered the volume of this space. This alteration in extracellular space was relatively small, and the quantitation of 45Ca movements was corrected for this change. The addition of 1 microM Bay K 8644 to the perfusate stimulated tension and augmented Ca2+ depletion from the extracellular space. The addition of 15 microM nifedipine to the perfusate significantly reduced both tension development and Ca2+ depletion from the extracellular space of the muscle. Net contraction-dependent movement of Ca2+ from the extracellular space into the myocardium under control conditions at 1 mM [Ca2+] was 10-14 mumol Ca2+/kg tissue wet wt/beat. This value indicates either a large contribution of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum and/or significant contribution of sarcolemmal bound Ca2+ to excitation-contraction coupling in the rabbit ventricle.     

Molecular coupling of a Ca2+-activated K+ channel to L-type Ca2+ channels via alpha-actinin2

Cytoskeletal proteins are known to sculpt the structural architecture of cells. However, their role as bridges linking the functional crosstalk of different ion channels is unknown. Here, we demonstrate that a small conductance Ca(2+)-activated K(+) channels (SK2 channel), present in a variety of cells, where they integrate changes in intracellular Ca(2+) concentration [Ca(2+)(i)] with changes in K(+) conductance and membrane potential, associate with L-type Ca(2+) channels; Ca(v)1.3 and Ca(v)1.2 through a physical bridge, alpha-actinin2 in cardiac myocytes. SK2 channels do not physically interact with L-type Ca(2+) channels, instead, the 2 channels colocalize via their interaction with alpha-actinin2 cytoskeletal protein. The association of SK2 channel with alpha-actinin2 localizes the channel to the entry of external Ca(2+) source, which regulate the channel function. Furthermore, we demonstrated that the functions of SK2 channels in atrial myocytes are critically dependent on the normal expression of Ca(v)1.3 Ca(2+) channels. Null deletion of Ca(v)1.3 channel results in abnormal function of SK2 channel and prolongation of repolarization and atrial arrhythmias. Our study provides insight into the molecular mechanisms of the coupling of SK2 channel with voltage-gated Ca(2+) channel, and represents the first report linking the coupling of 2 different types of ion channels via cytoskeletal proteins.     

Pharmacological basis of Ca2+ channels and Ca2+ channel antagonists

Ca2+ plays multiple roles in muscle E-C coupling, secretion, and neural transmission, in addition to survival, proliferation, and death of cells. The voltage-dependent L-type Ca2+ channel is a transmembrane protein that selectively permeates Ca2+ on activation by membrane depolarization. Ca2+ channel blockers (or Ca2+ antagonists) selectively block this channel. The blocking action is exerted in a tissue-specific manner, which underlies the unique pharmacological properties of Ca2+ channel blockers. The later generation of slowly-acting and long-lasting Ca2+ channel blockers has been designed to overcome the side effects of classical Ca2+ channel blockers. The pharmacological and molecular basis for the unique action of Ca2+ channel blockers will be discussed.     

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin

Ca2+-induced inhibition of alpha1C voltage-gated Ca2+ channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+ has been a matter of some controversy, as previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of alpha1C and/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Previously, using site directed mutagenesis, we have shown that disruption of the EF-hand motif does not remove Ca2+ inhibition. We now show that the IQ motif binds CaM and that disruption of this binding activity prevents Ca2+ inhibition. We propose that Ca2+ entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+ inhibition.     

Carbon monoxide dilates cerebral arterioles by enhancing the coupling of Ca2+ sparks to Ca2+-activated K+ channels

Carbon monoxide (CO) is generated endogenously by the enzyme heme oxygenase. Although CO is a known vasodilator, cellular signaling mechanisms are poorly understood and are a source of controversy. The goal of the present study was to investigate mechanisms of CO dilation in porcine cerebral arterioles. Data indicate that exogenous or endogenously produced CO is a potent activator of large-conductance Ca2+-activated K+ (K(Ca)) channels and Ca2+ spark-induced transient K(Ca) currents in arteriole smooth muscle cells. In contrast, CO is a relatively poor activator of Ca2+ sparks. To understand the apparent discrepancy between potent effects on transient K(Ca) currents and weak effects on Ca2+ sparks, regulation of the coupling relationship between these events by CO was investigated. CO increased the percentage of Ca2+ sparks that activated a transient K(Ca) current (ie, the coupling ratio) from approximately 62% in the control condition to 100% and elevated the slope of the amplitude correlation between these events approximately 2.6-fold, indicating that Ca2+ sparks induced larger amplitude transient K(Ca) currents in the presence of CO. This signaling pathway for CO is physiologically relevant because ryanodine, a ryanodine-sensitive Ca2+ release channel blocker that inhibits Ca2+ sparks, abolished CO dilation of pial arterioles in vivo. Thus, CO dilates cerebral arterioles by priming K(Ca) channels for activation by Ca2+ sparks. This study presents a novel dilatory signaling pathway for CO in the cerebral circulation and appears to be the first demonstration [corrected] of a vasodilator that acts by increasing the effective coupling of Ca2+ sparks to K(Ca) channels.     

Structure of the voltage-gated L-type Ca2+ channel by electron cryomicroscopy

Voltage-dependent L-type Ca(2+) channels play important functional roles in many excitable cells. We present a three-dimensional structure of an L-type Ca(2+) channel. Electron cryomicroscopy in conjunction with single-particle processing was used to determine a 30-A resolution structure of the channel protein. The asymmetrical channel structure consists of two major regions: a heart-shaped region connected at its widest end with a handle-shaped region. A molecular model is proposed for the arrangement of this skeletal muscle L-type Ca(2+) channel structure with respect to the sarcoplasmic reticulum Ca(2+)-release channel, the physical partner of the L-type channel for signal transduction during the excitation-contraction coupling in muscle.     

Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling

Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca(V)1.1 (alpha1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from alpha1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the beta-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR beta1a-subunit was mapped to the M(3201) to W(3661) region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K(3495)KKRR_ _R(3502), a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K(3495)KKRR_ _R(3502) cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca(2+) transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca(2+) content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca(2+) currents were slightly affected. The data show that the DHPR beta-subunit, like alpha1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca(2+) signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely alpha1S and beta1a, interacting with possibly different domains of RyR1.     

Ca2+ channel subtypes and pharmacology in the kidney

A large body of evidence has accrued indicating that voltage-gated Ca(2+) channel subtypes, including L-, T-, N-, and P/Q-type, are present within renal vascular and tubular tissues, and the blockade of these Ca(2+) channels produces diverse actions on renal microcirculation. Because nifedipine acts exclusively on L-type Ca(2+) channels, the observation that nifedipine predominantly dilates afferent arterioles implicates intrarenal heterogeneity in the distribution of L-type Ca(2+) channels and suggests that it potentially causes glomerular hypertension. In contrast, recently developed Ca(2+) channel blockers (CCBs), including mibefradil and efonidipine, exert blocking action on L-type and T-type Ca(2+) channels and elicit vasodilation of afferent and efferent arterioles, which suggests the presence of T-type Ca(2+) channels in both arterioles and the distinct impact on intraglomerular pressure. Recently, aldosterone has been established as an aggravating factor in kidney disease, and T-type Ca(2+) channels mediate aldosterone release as well as its effect on renal efferent arteriolar tone. Furthermore, T-type CCBs are reported to exert inhibitory action on inflammatory process and renin secretion. Similarly, N-type Ca(2+) channels are present in nerve terminals, and the inhibition of neurotransmitter release by N-type CCBs (eg, cilnidipine) elicits dilation of afferent and efferent arterioles and reduces glomerular pressure. Collectively, the kidney is endowed with a variety of Ca(2+) channel subtypes, and the inhibition of these channels by their specific CCBs leads to variable impact on renal microcirculation. Furthermore, multifaceted activity of CCBs on T- and N-type Ca(2+) channels may offer additive benefits through nonhemodynamic mechanisms in the progression of chronic kidney disease.     

Altered regulation of cardiac contraction and relaxation by Ca2+ in heart failure

Myocardial contractile dysfunction in congestive heart failure is characterized by a decrease in force developed and retardation of relaxation. These alterations are mainly due to those in intracellular Ca(2 +) transients (CaT) . CaT are regulated by a number of functional proteins, including sarcolemmal L-type Ca(2 +) channels, Na(+)/Ca(2 +) exchanger and Ca(2 +) ATPase, sarcoplasmic reticulum Ca(2 +) ATPase (SERCA 2 ) , phospholamban and ryanodine receptors, and mitochondrial Ca(2 +) uniporter. Changes in expression and function of these regulatory proteins that occur in the course of increasing severity of heart failure are responsible for the characteristic changes in force development and relaxation observed under pathophysiological conditions in congestive heart failure.     

Voltage dependence of cardiac excitation-contraction coupling: unitary Ca2+ current amplitude and open channel probability

Excitation-contraction coupling in cardiac myocytes occurs by Ca2+-induced Ca2+ release, where L-type Ca2+ current evokes a larger sarcoplasmic reticulum (SR) Ca2+ release. The Ca2+-induced Ca2+ release amplification factor or gain (SR Ca2+ release/I(Ca)) is usually assessed by the V(m) dependence of current and Ca2+ transients. Gain rises at negative V(m), as does single channel I(Ca) (i(Ca)), which has led to the suggestion that the increases of i(Ca) amplitude enhances gain at more negative V(m). However, I(Ca) = NP(o) x i(Ca) (where NP(o) is the number of open channels), and NP(o) and i(Ca) both depend on V(m). To assess how i(Ca) and NP(o) separately influence Ca2+-induced Ca2+ release, we measured I(Ca) and junctional SR Ca2+ release in voltage-clamped rat ventricular myocytes using "Ca2+ spikes" (confocal microscopy). To vary i(Ca) alone, we changed [Ca2+](o) rapidly at constant test V(m) (0 mV) or abruptly repolarized from +120 mV to different V(m) (at constant [Ca2+](o)). To vary NP(o) alone, we altered Ca2+ channel availability by varying holding V(m) (at constant test V(m)). Reducing either i(Ca) or NP(o) alone increased excitation-contraction coupling gain. Thus, increasing i(Ca) does not increase gain at progressively negative test V(m). Such enhanced gain depends on lower NP(o) and reduced redundant Ca2+ channel openings (per junction) and a consequently smaller denominator in the gain equation. Furthermore, modest i(Ca) (at V(m) = 0 mV) may still effectively trigger SR Ca2+ release, whereas at positive V(m) (and smaller i(Ca)), high and well-synchronized channel openings are required for efficient excitation-contraction coupling. At very positive V(m), reduced i(Ca) must explain reduced SR Ca2+ release.     

Cardiac function involving the T-type Ca2+ channel

Functional involvement of the T-type calcium channel in the heart excitation at the pathophysiological conditions has been elucidated. The T-type channel is classified as the low voltage-activated channel (LVA) solely in the voltage activated calcium channel. In 1998, Perez-Reyes and coworkers cloned the first LVA alpha(1) subunit, which was named as alpha(1G) or Ca(V)3.1. In the cardiac muscle, alpha(1G) and the other clone alpha(1H) are dominantly expressed in the sinoatrial node, atrioventricular node and other signal conduction tissues. Abnormal activity of T channels has been suggested in the following cardiovascular diseases: hypertension, cardiac hypertrophy, cardiac infarction. The cloning of the T-type calcium channel allows us to understand the function of the channel in detail and options for therapeutics in the T-type channel-related diseases.     

Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation

The cardiac SR Ca(2+)-ATPase (SERCA2a) regulates intracellular Ca(2+)-handling and thus, plays a crucial role in initiating cardiac contraction and relaxation. SERCA2a may be modulated through its accessory phosphoprotein phospholamban or by direct phosphorylation through Ca(2+)/calmodulin dependent protein kinase II (CaMK II). As an inhibitory component phospholamban, in its dephosphorylated form, inhibits the Ca(2+)-dependent SERCA2a function, while protein kinase A dependent phosphorylation of the phospho-residues serine-16 or Ca(2+)/calmodulin-dependent phosphorylation of threonine-17 relieves this inhibition. Recent evidence suggests that direct phosphorylation at residue serine-38 in SERCA2a activates enzyme function and enhances Ca(2+)-reuptake into the sarcoplasmic reticulum (SR). These effects that are mediated through phosphorylation result in an overall increased SR Ca(2+)-load and enhanced contractility. In human heart failure patients, as well as animal models with induced heart failure, these modulations are altered and may result in an attenuated SR Ca(2+)-storage and modulated contractility. It is also believed that abnormalities in Ca(2+)-cycling are responsible for blunting the frequency potentiation of contractile force in the failing human heart. Advanced gene expression and modulatory approaches have focused on enhancing SERCA2a function via overexpressing SERCA2a under physiological and pathophysiological conditions to restore cardiac function, cardiac energetics and survival rate.     

K+ and Ca2+ channel activity and cytosolic [Ca2+] in oxygen-sensing tissues.

Ion channels are known to participate in the secretory or mechanical responses of chemoreceptor cells to changes in oxygen tension (P(O2)). We review here the modifications of K+ and Ca2+ channel activity and the resulting changes in cytosolic [Ca2+] induced by low P(O2) in glomus cells and arterial smooth muscle which are well known examples of O2-sensitive cells. Glomus cells of the carotid body behave as presynaptic-like elements where hypoxia produces a reduction of K+ conductance leading to enhanced membrane excitability, Ca2+ entry and release of dopamine and other neurotransmitters. In arterial myocytes, hypoxia can inhibit or potentiate Ca2+ channel activity, thus regulating cytosolic [Ca2+] and contraction. Ca2+ channel inhibition is observed in systemic myocytes and most conduit pulmonary myocytes, whereas potentiation is seen in a population of resistance pulmonary myocytes. The mechanism whereby O2 modulates ion channel activity could depend on either the direct allosteric modulation by O2-sensing molecules or redox modification by reactive chemical species.     

Ionic dependence of Ca2+ channel modulation by syntaxin 1A

Alteration of the kinetic properties of voltage-gated Ca(2+) channels, Ca(v)1.2 (Lc-type), Ca(v)2.2 (N type), and Ca(v)2.3 (R type), by syntaxin 1A (Syn1A) and synaptotagmin could modulate exocytosis. We tested how switching divalent charge carriers from Ca(2+) to Sr(2+) and Ba(2+) affected Syn1A and synaptotagmin modulation of Ca(2+)-channel activation. Syn1A accelerated Ca(v)1.2 activation if Ca(2+) was the charge carrier; and by substituting for Ba(2+), Syn1A slowed Ca(v)1.2 activation. Syn1A also significantly accelerated Ca(v)2.3 activation in Ca(2+) and marginally in Ba(2+). Synaptotagmin, on the other hand, increased the rate of activation of Ca(v)2.3 and Ca(v)2.2 in all permeating ions tested. The Syn1A-channel interaction, unlike the synaptotagmin-channel interaction, proved significantly more sensitive to the type of permeating ion. It is well established that exocytosis is affected by switching the charge carriers. Based on the present results, we suggest that the channel-Syn1A interaction could respond to the conformational changes induced within the channel during membrane depolarization and divalent ion binding. These changes could partially account for the charge specificity of synaptic transmission as well as for the fast signaling between the Ca(2+) source and the fusion apparatus of channel-associated-vesicles (CAV). Furthermore, propagation of conformational changes induced by the divalent ions appear to affect the concerted interaction of the channel with the fusion/docking machinery upstream to free Ca(2+) buildup and/or binding to a cytosolic Ca(2+) sensor. These results raise the intriguing possibility that the channel is the Ca(2+) sensor in the process of fast neurotransmitter release.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Phosphorylation and dephosphorylation of dihydropyridine-sensitive voltage-dependent Ca2+ channel in skeletal muscle membranes by cAMP- and Ca2+-dependent processes.

The phosphorylation and dephosphorylation of the dihydropyridine-sensitive Ca2+ channel was studied in transverse-tubule membranes isolated from rabbit skeletal muscle. Exposure of these membranes to either the cAMP-dependent protein kinase or a Ca2+/calmodulin-dependent protein kinase resulted in a rapid phosphorylation of a protein with properties similar to the major component of the skeletal muscle Ca2+ channel. The molecular mass of the phosphoprotein was 140 or 160 kDa, depending on the electrophoretic conditions. The stoichiometry of the phosphorylation was calculated to be 0.4-1.0 mol of phosphate per mol of protein. Neither the rate nor the extent of phosphorylation was affected by dihydropyridines. Limited proteolytic digestion of the protein that had been phosphorylated by either or both protein kinases yielded a single phosphopeptide of approximately equal to 5.4 kDa. The Ca2+-dependent phosphatase calcineurin dephosphorylated the membrane-bound Ca2+ channel that had been previously phosphorylated by either protein kinase. The results suggest that the major component of the dihydropyridine-sensitive Ca2+ channel from skeletal muscle can be effectively phosphorylated and dephosphorylated in its native state by cAMP- and Ca2+-dependent processes.