The Local Control of Cytosolic Ca2+ as a Propagator of CNS Communication—Integration of Mitochondrial Transport Mechanisms and Cellular Responses

Ca²⁺ signals propagate in wave form along individual cells of the central nervous system(CNS) and through networks of connected cells of neuronal and multiple glial cell types. Inorder for wave fronts to convey information, signaling mechanisms are required that allowwaves to propagate reproducibly and without decrement in signal strength over long distances.CNS Ca²⁺ waves are under specific integrated local control, made possible by interactions atlocal subcellular microdomains between endoplasmic reticulum and mitochondria. Activemitochondria located near the mouth of inositol trisphosphate receptor (InsP₃R) channel clustersin glia take up Ca²⁺, which may prevent a buildup of Ca²⁺ around the InsP₃R channel, therebydecreasing the rate of Ca²⁺-induced receptor inactivation, and prolonging channel open time.Mitochondria may amplify InsP;i3-dependent Ca2;pl signals by a transient permeability transitionin response to Ca²⁺ uptake into the mitochondrion. Other evidence suggests privileged accessinto mitochondria for Ca²⁺ entering neurons by glutamatergic receptor channels. This enablesspecific signal modulation as the Ca²⁺ wave is propagated into neurons, such that mitochondrialocated close to glutamate channels can prolong the neuronal cytosolic response time bysuccessive uptake and release of Ca²⁺. Disruption of mitochondrial function deregulates theability of CNS-derived cells to undergo normal Ca²⁺ signaling and wave propagation.     

Activation of Barnea candida (mollusca,pelecypoda) oocytes by sperm or KCl,but not by NH4Cl,requires a calcium influx

Artificial activation of Barnea candida oocytes by NH₄Cl and the addition of excess KCl are, respectively, independent and dependent upon external Ca²⁺ concentration. The activating efficiency of NH₄Cl increases when external pH is raised which suggests that the proportion of unionized penetrating NH₃ is the key factor for activation under these conditions. The external Ca²⁺-dependent period for KCl or sperm-induced activation does not exceed 3–4 min. The transmembrane Ca²⁺ flux inhibitor D-600 reversibly inhibits KCl- or sperm-induced activation and the D-600-sensitive period closely corresponds to the Ca²⁺-dependent period. Ca²⁺ ions alone can also trigger activation provided external Mg²⁺ concentration is lowered. Finally, a direct demonstration is presented for the existence of a D-600-sensitive Ca²⁺ uptake following KCl- or sperm-induced activation, by using the radioactive tracer ⁴⁵Ca. The significance of that Ca²⁺ influx is discussed with respect to the possible involvement of an intracellular pH shift as a main factor in the activation of lamellibranch mollusc oocytes.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase

Experiments were performedto determine whether the organic Ca²⁺ channel blocker D-600(gallopamil), which penetrates into muscle cells, affects sarcoplasmicreticulum (SR) Ca²⁺ uptake by directly inhibiting the lightSR Ca²⁺-ATPase. We have previously shown that at 10 µM,D-600 inhibits LSR ATP-dependent Ca²⁺ uptake by 50% buthas no effect on ATPase activity (21). These data suggestthat the SR Ca²⁺-ATPase might be a potential target forD-600. The ATPase activity of the enzyme is associated with itshydrophilic cytoplasmic domain, whereas Ca²⁺ binding andtranslocation are associated with the transmembrane domain(18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermalinactivation experiments using the SR Ca²⁺-ATPasedemonstrated that D-600 decreased the thermal stability ofCa²⁺ transport but had no effect on the stability of ATPaseactivity. In addition, D-600 at a concentration of 160 µM did nothave any leaking effect of Ca²⁺ on theCa²⁺-loaded SR. Thermal denaturation profiles of SRmembranes revealed that D-600 interacts directly with the transmembranedomain of the Ca²⁺-ATPase. No evidence for interaction withthe nucleotide domain was obtained. We conclude that theCa²⁺ blocker D-600 inhibits the SR Ca²⁺ pumpspecifically by interacting with the transmembraneCa²⁺-binding domain of the Ca²⁺-ATPase.

     

Mechanistic distinction between activation and inhibition of (Na+K)-ATPase-mediated Ca influx in cardiomyocytes

(Na⁺+K⁺)-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na⁺/Ca²⁺-exchanger (NCX) plays a critical role in increasing intracellular Ca²⁺ concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on ⁴⁵Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced ⁴⁵Ca influx, suggesting that the Ca²⁺ influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca²⁺ channel (LTCC) inhibitor, completely blocks the activation of NKA-induced ⁴⁵Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca²⁺. In contrast, the inhibition of NKA by ouabain induces 4.7-fold ⁴⁵Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced ⁴⁵Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca²⁺ and that the NCX reverse-mode is the major source for the ⁴⁵Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca²⁺ increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca²⁺ influx path ways in cardiomyocytes.     

Calcium-activated chloride fluxes in cultured NCL-SG3 sweat gland cells

The dependence of chloride permeability of the human sweat gland cell line NCL-SG3 cell line on cytosolic free calcium ([Ca²⁺]_i) was investigated. X-ray microanalysis, fura-2 fluorescence and patch clamp methodology were used. Carbachol and A23187 decreased cellular Cl and K for cells grown on permeable supports, but carbachol had no effect on cells grown on impermeable supports. In perforated patch experiments with impermeable supports, ATP and calcium ionophores increased the inward current (ic) whereas carbachol had no effect. ic was unaffected by cation channel blockers or removal of extracellular Na⁺ but was blocked by chloride channel blockers. Lowering bath Ca²⁺ decreased ic. On raising bath Ca²⁺ ic and [Ca²⁺]_i responded with a transient rise which was not blocked by La³⁺ or D-600. La³⁺, but not D-600, blocked the entry of Mn²⁺. K⁺-depolarization and Bay-K-8644 had little effect on [Ca²⁺]_i. The rise in [Ca²⁺]_i may be mediated primarily via depletion operated Ca²⁺-channels. Irrespective of substrate NCL-SG3 cells have a chloride permeability which depends on [Ca²⁺]_i.     

Cytoplasmic calcium affects the gating of potassium channels in the plasma membrane ofChara corallina: a whole-cell study using calcium-channel effectors

The action of a wide range of drugs effective on Ca²⁺ channels in animal tissues has been measured on Ca²⁺ channels open during the action potential of the giant-celled green alga,Chara corallina. Of the organic effectors used, only the 1,4-dihydropyridines were found to inhibit reversibly Ca²⁺ influx, including, unexpectedly, Bay K 8644 and both isomers of 202–791. Methoxyverapamil (D-600), diltiazem, and the diphenylbutylpiperidines, fluspirilene and pimozide were found not to affect the Ca²⁺ influx. Conversely, bepridil greatly and irreversibly stimulated Ca²⁺ influx, and with time, stopped cytoplasmic streaming (which is sensitive to increases in cytoplasmic Ca²⁺). By apparently altering the cytoplasmic Ca²⁺ levels with various drugs, it was found that (with the exception of the inorganic cation, La³⁺) treatments likely to lead to an increase in cytoplasmic Ca²⁺ levels caused an increase in the rate of closure of the K⁺ channels. Similarly, treatments likely to lead to a decrease in cytoplasmic Ca²⁺ decreased the rate of K⁺ channel closure. The main effect of bepridil on the K⁺ channels was to increase the rate of voltage-dependent channel closure. The same effect was obtained upon increasing the external concentration of Ca²⁺, but it is likely that this was due to effects on the external face of the K⁺ channel. Addition of any of the 1,4-dihydropyridines had the opposite effect on the K⁺ channels, slowing the rate of channel closure. They sometimes also reduced K⁺ conductance, but this could well be a direct effect on the K⁺ channel; high concentrations (50 to 100 μM) of bepridil also reduced K⁺ conductance. No effect of photon irradiance or of abscisic acid could be consistently shown on the K⁺ channels. These results indicate a control of the gating of K⁺ channels by cytoplasmic Ca²⁺, with increased free Ca²⁺ levels leading to an increased rate of K⁺-channel closure. As well as inhibiting Ca²⁺ channels, it is suggested that La³⁺ acts on a Ca²⁺-binding site of the K⁺ channel, mimicking the effect of Ca²⁺ and increasing the rate of channel closure.     

Activation of Ca2+/Mg2+ ATPase in heart sarcolemma upon electrical stimulation

Electrical stimulation of the rat heart sarcolemmal membranes with a square wave current was found to increase Ca²⁺-ATPase activity. This activation of the enzyme was dependent upon the voltage of the electric current, frequency of stimulation and duration of stimulation of the sarcolemmal membranes. The increase in ca²⁺-ATPase was reversible upon terminating the electrical stimulation. The activation of sarcolemmal Ca²⁺-ATPase due to electrical stimulation was markedly depressed when the reaction was carried out at high pH (7.8 to 8.2), low pH (6.6 to 7.0), high temperatures (45 to 50°C) and low temperatures (17 to 25°C) of the incubation medium. Ca²⁺-antagonists, verapamil and D-600, unlike other types of inhibitors such as propranolol and ouabain, were found to reduce the activation of sarcolemmal Ca²⁺-ATPase by electrical stimulation. These results support the view that Ca²⁺/Mg²⁺ ATPase may be involved in the gating mechanism for opening Ca²⁺-channels in the sarcolemmal membrane upon excitation of the cardiac muscle.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Summary Smooth muscle cells normally do not possess fast Na²⁺ channels, but inward current is carried through two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]₀, and was inhibited by TTX (K_0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]₀, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na²⁺ channel current, and that the slow inward current is a Ca²⁺ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na⁺ channels and dihudropuridine-sensitive slow Ca²⁺ channels. The number of fast Na⁺ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na²⁺ channels, and it suggested that the fast Na⁺ current may be involved in spread of excitation. The Ca²⁺ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na⁺ channels and Ca²⁺ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either I_Ca(s) or I_Na(f), whereas Mg²⁺ (K_0.5 of 12 mM) and nifedipine (K_0.5 of 3.3 nM) depressed I_Ca(s). Oxytocin had no effect on I_Na(f) and actually depressed I_Ca(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of I_Ca(s), whereas that of Mg²⁺ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of I_Ca(s).     

Calcium Permeability of Non-N-Methyl-D-Aspartate Receptor Channels in Immature Cerebellar Purkinje Cells: Studies Using Fura-2 Microfluorometry

Abstract: Using fura-2 microfluorometry, I investigated the mechanism by which non-N-methyl-d -aspartate (NMDA) receptor agonists increase the cytosolic free calcium concentration ([Ca]_in) in single cerebellar Purkinje cells isolated from 3–10-day-old rats. Kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate dose-dependently increased the cytosolic free Na⁺ concentration, which was measured using sodium-binding benzofuran isophthalate microfluorometry, confirming the Na⁺ influx through ion channels linked to non-NMDA receptors. The [Ca²⁺] increases induced by relatively lower concentrations of agonists were entirely dependent on external Ca²⁺ and were reduced by removal of external Na⁺ or by addition of a Ca²⁺ channel blocker, D600. The results indicate that the non-NMDA agonist–induced [Ca]_in increase was due mainly to Ca²⁺ influx through voltage-dependent Ca²⁺ channels, which were activated by a massive Na⁺ influx. On the other hand, higher concentrations of agonists dose-dependently increased [Ca]_in under conditions in which activation of voltage-dependent Ca²⁺ channels were blocked by a combination of Na⁺ removal with D600. These [Ca]_in increases were Ca²⁺ dependent and little affected by adding a competitive NMDA antagonist. Non-NMDA agonists also stimulated influxes of Mn²⁺ and Co²⁺, both of which can be monitored by quenching fura-2 fluorescence under the same conditions. These results suggest that ion channels linked to non-NMDA receptors on immature Purkinje cells are permeable to Ca²⁺, Mn²⁺, and Co²⁺.     

Comparison of the Effects Exerted by Luminal Ca2+ on the Sensitivity of the Cardiac Ryanodine Receptor to Caffeine and Cytosolic Ca2+

Ca²⁺ released from the sarcoplasmic reticulum (SR) via ryanodine receptor type 2 (RYR2) is the key determinant of cardiac contractility. Although activity of RYR2 channels is primary controlled by Ca²⁺ entry through the plasma membrane, there is growing evidence that Ca²⁺ in the lumen of the SR can also be effectively involved in the regulation of RYR2 channel function. In the present study, we investigated the effect of luminal Ca²⁺ on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca²⁺ added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba²⁺ or Ca²⁺. The given ion served also as a charge carrier. Luminal Ca²⁺ effectively shifted the EC₅₀ for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca²⁺. Importantly, luminal Ca²⁺ exerted an effect on channel gating kinetics. Both the open and closed dwell times were considerably prolonged over the whole range (response to caffeine) or the partial range (response to cytosolic Ca²⁺) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca²⁺ with the luminally located Ca²⁺ regulatory sites on the RYR2 channel complex.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

Regulation of bi-directional communication between intracellular Ca²⁺ pools and surface Ca²⁺ channels remains incompletely characterized. We report Ca²⁺ release mediated by inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca²⁺ release, Ca²⁺ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP₃R induced Ca²⁺ release, whereas RyR-mediated Ca²⁺ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP₃R and RyR linked Ca²⁺ influx even though Ca²⁺ pool sensitivities were different. These findings suggest discrete Ca²⁺ store/Ca²⁺ channel coupling mechanisms in the IP₃R and RyR pathways as revealed by the differential sensitivity to actin perturbation.     

Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum

In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K⁺), and the presence of short and long lasting subconducting states. Its open probability P_o increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca²⁺ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K⁺ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca²⁺] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca²⁺, Ba²⁺ or Mg²⁺-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by µM Ca²⁺ while its Ca²⁺ sensitivity was potentiated by millimolar ATP. However Mg²⁺ and calmodulin modulated its gating behavior. Ca²⁺ releasing drugs such as caffeine and ryanodine increased its P_o. All these features are characteristics of the SR Ca²⁺ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway. This channel displays all the regulatory sites of the native cardiac SR Ca²⁺ release channel. However, when NA was used as charge carrier, multiple subconducting states were observed. In conclusion, the reconstitution experiments have yield a great deal of informations about the biochemical and biophysical events that may regulated the ionic flux across the SR membrane.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Methamphetamine directly accelerates beating rate in cardiomyocytes by increasing Ca entry via L-type Ca channel

Methamphetamine induces several cardiac dysfunctions, which leads to arrhythmia, cardiac failure and sudden cardiac death. Although these cardiac alterations elicited by methamphetamine were thought to be due to an indirect action of methamphetamine, namely, an excessive catecholamine release from synaptic terminals, while it seems likely that methamphetamine directly modulates the functioning of cardiomyocytes independent of neurotransmitters. However, the direct effects of methamphetamine on cardiomyocytes are still not clear. We show that methamphetamine directly accelerates the beating rate and alters Ca²⁺ oscillation pattern in cultured neonatal rat cardiomyocytes. Adrenergic receptor antagonists did not block the methamphetamine-induced alterations in cardiomyocytes. Treatment with a ryanodine receptor type 2 inhibitor and a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor did not affect these responses, either. In contrast, the L-type Ca²⁺ channel inhibitor nifedipine eradicated these responses. Furthermore, methamphetamine elevated the internal free Ca²⁺ concentration in HEK-293T cells stably transfected with the L-type Ca²⁺ channel α1C subunit. In neonatal rat cardiomyocytes, methamphetamine accelerates beating rate and alters Ca²⁺ oscillation pattern by increasing Ca²⁺ entry via the L-type Ca²⁺ channels independent of any neurotransmitters.     

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca²⁺ via activation of the steady-state voltage dependent R-type Ca²⁺ channels, endothelin-1 (10^-7 M) and insulin (80 U/ml) were found to induce a sustained increase in cytosolic free Ca²⁺ ([Ca]_i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca²⁺.However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca²⁺ influx and [Ca]_i elevation via activation of the R-type Ca²⁺ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca²⁺ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca²⁺ channel blocker, nifedipine, but were completely reversed by the R-type Ca²⁺ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca²⁺ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).     

Mitochondrial calcium channels

Mitochondrial Ca²⁺ handling plays an important role in energy production and various cellular signaling processes. Mitochondrial Ca²⁺ uptake is regulated by the mitochondrial Ca²⁺ uniporter (MCU), at least one non-MCU Ca²⁺ channel and possibly a mitochondrial ryanodine receptor. Two distinct mechanisms mediate Ca²⁺ outward transport, the Na⁺-dependent (mNCX) and the Na⁺-independent Ca²⁺ efflux. In recent years we gained more insight into the regulation and function of these different Ca²⁺ transport mechanisms. However, the precise physiological role and the molecular structure of all mitochondrial Ca²⁺ transporters and channels still has to be determined.