Effect of ryanodine on cardiac calcium current and calcium channel gating current.

The effects of 100 microM ryanodine on the L-type calcium channel were studied using the pacth-clamp technique in isolated guinea pig ventricular myocytes. The inactivation kinetics of the calcium current were slowed down in the presence of ryanodine in agreement with the blockade of the release of calcium from the sarcoplasmic reticulum by the drug. The I-V and steady-state inactivation curves of the calcium current were shifted to negative values by ryanodine. A similar shift was observed in the activation and inactivation curves of the intramembrane charge movement associated with the calcium channel. Due to this shift, ryanodine slightly reduced the maximal amount of displaced charge although it did not modify the transition from the inactivated to the activated state (i.e., charge movement repriming). This result is in notable contrast with that obtained in skeletal muscle, where it has been found that ryanodine interferes with charge movement repriming. These results provide additional evidence of the postulated differences between the architecture of the excitation-contraction coupling system in cardiac and skeletal muscle.     

Photoalteration of calcium channel blockade in the cardiac Purkinje fiber.

Organic compounds that block calcium channel current (calcium antagonists) are important tools for the characterization of this channel. However, the practically irreversible nature of this block restricts the usefulness of this group of drugs. In this paper, we investigate the influence of light on calcium channel blockade by several organic compounds. Our results show that inhibition of calcium channel current by two dihydropyridine derivatives that contain an o-nitro moiety (nisoldipine and nifedipine) can be rapidly reversed by illumination. The energy range important to this reaction is for light wavelengths between 320 and 450 nm. Calcium channel inhibition by two other dihydropyridine derivatives (nicardipine and nitrendipine) as well as by D600, is not modulated by illumination. These results indicate that the photosensitivity of certain dihydropyridine calcium channel blockers make these compounds useful as reversible blockers of this channel.     

Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel.

The skeletal muscle dihydropyridine receptor/Ca2+ channel is composed of five protein components (alpha 1, alpha 2 delta, beta, and gamma). Only two such components, alpha 1 and alpha 2, have been identified in heart. The present study reports the cloning and expression of a novel beta gene that is expressed in heart, lung, and brain. Coexpression of this beta with a cardiac alpha 1 in Xenopus oocytes causes the following changes in Ca2+ channel activity: it increases peak currents, accelerates activation kinetics, and shifts the current-voltage relationship toward more hyperpolarized potentials. It also increases dihydropyridine binding to alpha 1 in COS cells. These results indicate that the cardiac L-type Ca2+ channel has a similar subunit structure as in skeletal muscle, and provides evidence for the modulatory role of the beta subunit.     

A minimal gating model for the cardiac calcium release channel.

A Markovian model of the cardiac Ca release channel, based on experimental single-channel gating data, was constructed to understand the transient nature of Ca release. The rate constants for a minimal gating scheme with one Ca-free resting state, and with two open and three closed states with one bound Ca2+, were optimized to simulate the following experimental findings. In steady state the channel displays three modes of activity: inactivated 1 mode without openings, low-activity L mode with single openings, and high-activity H mode with bursts of openings. At the onset of a Ca2+ step, the channel first activates in H mode and then slowly relaxes to a mixture of all three modes, the distribution of which depends on the new Ca2+. The corresponding ensemble current shows rapid activation, which is followed by a slow partial inactivation. The transient reactivation of the channel (increment detection) in response to successive additions of Ca2+ is then explained by the model as a gradual recruitment of channels from the extant pool of channels in the resting state. For channels in a living cell, the model predicts a high level of peak activation, a high extent of inactivation, and rapid deactivation, which could underlie the observed characteristics of the elementary release events (calcium sparks).     

Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel.

Purified canine cardiac sarcoplasmic reticulum vesicles were passively loaded with 45CaCl2 and assayed for Ca2+ releasing activity according to a rapid quench protocol. Ca2+ release from a subpopulation of vesicles was found to be activated by micromolar Ca2+ and millimolar adenine nucleotides, and inhibited by millimolar Mg2+ and micromolar ruthenium red. 45Ca2+ release in the presence of 10 microM free Ca2+ gave a half-time for efflux of 20 ms. Addition of 5 mM ATP to 10 microM free Ca2+ increased efflux twofold (t1/2 = 10 ms). A high-conductance calcium-conducting channel was incorporated into planar lipid bilayers from the purified cardiac sarcoplasmic reticulum fractions. The channel displayed a unitary conductance of 75 +/- 3 pS in 53 mM trans Ca2+ and was selective for Ca2+ vs. Tris+ by a ratio of 8.74. The channel was dependent on cis Ca2+ for activity and was also stimulated by millimolar ATP. Micromolar ruthenium red and millimolar Mg2+ were inhibitory, and reduced open probability in single-channel recordings. These studies suggest that cardiac sarcoplasmic reticulum contains a high-conductance Ca2+ channel that releases Ca2+ with rates significant to excitation-contraction coupling.     

Regulation of the cardiac calcium channel by protein phosphatases

The calcium current (ICa) through the L-type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285-293]. We investigated the possible contribution of the 'catalytic subunits' of protein phosphatase 1 and 2A in the down-regulation of the cardiac calcium channel. Single guinea-pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 microM) as well as 2A (2.3 microM) completely abolished the increase of ICa induced by isoprenaline (0.05 microM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor-2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash-out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor-2. (4) Perfusion of the myocyte under basal conditions with adenosine 5'-[gamma-thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 microM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min. From these results we make the following conclusions. (1) The calcium channel from guinea-pig myocytes is regulated by phosphorylation-dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down-regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.     

Calsequestrin and the calcium release channel of skeletal and cardiac muscle

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.     

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.     

High affinity binding of a calcium channel antagonist to smooth and cardiac muscle

Specific binding of the Ca²⁺-channel antagonist nitrendipine, a close structural analog of nifedipine, has been measured in microsomal membrane fractions from guinea pig ileal longitudinal smooth muscle. The dissociation constant was 0.18 nanomole per liter and maximum binding was 1.14 picomoles per milligram of protein. Binding with very similar characteristics was found in a rat ventricle preparation. This high affinity binding was sensitive to displacement by a series of 1,4-dihydropyridine analogs of nifedipine with an activity sequence correlating well with that determined for inhibition of mechanical responses in the intact smooth muscle.     

Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity.

Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+ release channel by CaM kinase may provide a unique mechanism for regulating intracellular Ca2+ release.     

The role of region IVS5 of the human cardiac calcium channel in establishing inactivated channel conformation: use-dependent block by benzothiazepines

The role of inactivated channel conformation and use dependence for diltiazem, a specific benzothiazepine calcium channel inhibitor, was studied in chimeric constructs and point mutants created in the IVS5 transmembrane segment of the L-type cardiac calcium channel. All mutations, chimeric or point mutations, were restricted to IVS5, while the YAI-containing segment in IVS6, i.e. the primary interaction site with benzothiazepines, remained intact. Slowed inactivation rate and incomplete steady state inactivation, a behavior of some mutants, were accompanied by a reduced or by a complete loss of use-dependent block by diltiazem. Single channel properties of mutants that lost use dependence toward diltiazem were characterized by drastically elongated mean open times and distinctly slower time constants of open time distribution. Mutation of individual residues of the IVMLF segment in IVS5 did not mimic the complete loss of use dependence as observed for the replacement of the whole stretch. These results establish evidence that amino acids that govern inactivation and the drug-binding site and other amino acids that are located distal from the putative drug-binding site contribute significantly to the function of the benzothiazepine receptor region. The data are consistent with a complex "pocket" conformation that is responsive to a specific class of L-type calcium channel inhibitors. The data allow for a concept that multiple sites within regions of the alpha(1) subunit contribute to auto-regulation of the L-type Ca(2+) channel.     

Purification and characterization of the dihydropyridine-sensitive voltage-dependent calcium channel from cardiac tissue

The dihydropyridine-sensitive voltage-dependent Ca2+ channel from cardiac tissue was purified 900-fold using DEAE-Sephadex A-25, concanavalin A-Sepharose, and wheat germ agglutinin-Sepharose. The purified preparation was highly enriched in a peptide of 140,000 daltons when electrophoresed on sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol, or 170,000 when electrophoresed in the presence of iodoacetamide. Polyclonal antibodies raised against the purified subunits of the rabbit skeletal muscle Ca2+ channel recognized the 170-kDa protein in preparations electrophoresed under nonreducing conditions, and the large peptide of 140 kDa and smaller peptides of 29-32 kDa in preparations analyzed under reducing conditions. Monoclonal antibodies, which were raised against the native Ca2+ channel from skeletal muscle, immunoprecipitated [3H]PN 200-110 binding activity from solubilized cardiac membranes and immunoprecipitated 125I-labeled peptides (from the purified cardiac Ca2+ channel preparation) which migrated as a single species of 170 kDa under nonreducing conditions, or as 140, 32, and 29 kDa under reducing conditions. The results show that the purified cardiac Ca2+ channel, like that previously purified from skeletal muscle, consists of a major component of 170 kDa which is comprised of a 140-kDa peptide linked by disulfide bonds to smaller peptides of 32-29 kDa. Peptide maps of the 140-kDa peptide purified from cardiac and skeletal muscle preparations were strikingly similar, suggesting a high degree of homology in their primary sequence.     

Elevated myocardial calcium and its role in sudden cardiac death.

The contribution of intracellular calcium to ventricular fibrillation (VF) was investigated using chronically instrumented dogs with healed myocardial infarctions. A 2-minute coronary occlusion was initiated during the last minute of exercise. Fourteen animals developed ventricular fibrillation (susceptible) whereas the remaining 12 did not (resistant) during this exercise plus ischemia test. The test was then repeated for the susceptible animals after pretreatment with the intracellular calcium chelator BAPTA-AM (1.0 mg/kg). BAPTA-AM significantly reduced left ventricular dp/dt max and prevented VF in 8 of 12 susceptible animals. Conversely, myocardial cytosolic calcium levels were increased in resistant animals using the calcium channel agonist Bay K 8644 (30 micrograms/kg) or phenylephrine (10 micrograms.kg-1.min-1 3-5 min before occlusion). Bay K 8644 induced VF in all 5 resistant animals tested whereas phenylephrine induced VF in 8 of 12 resistant animals. BAPTA-AM pretreatment attenuated the hemodynamic effects of Bay K 8644 or phenylephrine and prevented VF in five of five Bay K 8644- and four of seven phenylephrine-treated animals. Finally, the endogenous level of calcium/calmodulin (Ca-CaM)-dependent phosphorylation of 170- and 55-kDa substrate proteins was measured (as an index of intracellular free calcium concentration). In the susceptible dog heart, the endogenous level of Ca-CaM-dependent phosphorylation was estimated to be two- to threefold higher than that observed in resistant dog heart. Treatment of resistant dog tissue with the calcium ionophore A23187 increased the level of Ca-CaM-dependent phosphorylation of these two proteins to the level observed in susceptible dog heart. These data suggest that elevated cytosolic calcium facilitates development of malignant arrhythmias and that elevated cytosolic calcium levels may be present in animals particularly susceptible to ventricular fibrillation.     

Description of modal gating of the cardiac calcium release channel in planar lipid membranes.

Single channel activity of the cardiac ryanodine-sensitive calcium-release channel in planar lipid membranes was studied in order to elucidate the calcium-dependent mechanism of its steady-state behavior. The single channel kinetics, observed with Cs+ as the charge carrier at different activating (cis) Ca2+ concentrations in the absence of ATP and Mg2+, were similar to earlier reports and were extended by analysis of channel modal behavior. The channel displayed three episodic levels of open probability defining three gating modes: H (high activity), L (low activity), and I (no activity). The large difference in open probabilities between the two active modes resulted from different bursting patterns and different proportions of two distinct channel open states. I-mode was without openings and can be regarded as the inactivated mode of the channel; L-mode was composed of short and sparse openings; and H-mode openings were longer and grouped into bursts. Modal gating may explain calcium-release channel adaptation (as transient prevalence of H-mode after Ca2+ binding) and the inhibitory effects of drugs (as stabilization of mode I), and it provides a basis for understanding the regulation of calcium release.     

The calcium channel: electrophysiological findings in cardiac cells

General properties of the calcium channel are analyzed in the myocardium under voltage clamp conditions both in multicellular properties and in single isolated cells. More recently the patch-clamp has allowed us to study single channels. In normal conditions, the selectivity of the calcium channel to Ca2+ ions is very high; however, in the absence of calcium many divalent cations and even Na ions can go through this channel. Kinetic analysis shows: calcium channel inactivation depends on Ca2+ entry rather than on membrane potential, opening of this channel requires at least two transitions of closed states before the open state. Many works refer to pharmacology of the calcium channel in heart tissue. beta-adrenergic stimulation induces a large increase in current amplitude related to the increase in maximal conductance without variation in the unitary conductance. Two interpretations are available: an increase in the opening probability and/or an increase in the number of available channels, both are consecutives to phosphorylations of the channel. Cholinergic stimulation seems to have little effect. Studies of calcium antagonists have revealed that all these substances have, at various levels, use-dependent and voltage-dependent inhibitory effect. Moreover, some dihydropyridine derivatives can even, activate the channel. Antiarrhythmic as well as general anaesthetic agents have an inhibitory action on the calcium channel besides their effects on the sodium channel or the Na-Ca exchange. Very recently, the existence of another calcium conductance was demonstrated. It is characterized by a low threshold, a pure voltage-dependent inactivation, a relatively weak sensitivity to anticalcic agents and neurotransmettors.     

Ultrastructural localization of calcium around the membrane of the surface connected system in the human platelet.

The localization of calcium in the membrane system of human platelets was determined by ultrahistochemical methods equipped with an electron probe x-ray microanalyzer. After potassium oxalate-glutaraldehyde treatment large amounts of electron opaque precipitates were observed around the membrane of the surface connected system. Electron probe x-ray microanalysis clearly defined that the precipitates were composed of calcium oxalate. The localization of calcium on the membrane of the surface connected system was also confirmed even after treatment of the platelets with potassium antimonate-OsO4. These results support a model which depicts the surface connected membrane system taking part in the store and the transport of calcium.     

Reconstruction of the dihydropyridine site in a non-L-type calcium channel: the role of the IS6 segment.

Mutations of eight to nine amino acids of IIIS5, IIIS6 and IVS6 segments were shown to reconstruct the dihydropyridine (DHP) interaction site in the non-L-type alpha1E or alpha1A calcium channels. The reconstructed site enabled enantiomer-selective inhibition and activation of the expressed chimeras by DHPs but failed to transfer voltage dependence of the current inhibition. Here we show that transfer of four non-conserved amino acids from the IS6 segment to the DHP-sensitive alpha1E chimera increased the inhibition by (+)isradipine at the hyperpolarized membrane potential of -100 mV and enhanced the voltage-dependent block.     

Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance

Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel.