Saltatory propagation of Ca2+ waves by Ca2+ sparks.

Punctate releases of Ca2+, called Ca2+ sparks, originate at the regular array of t-tubules in cardiac myocytes and skeletal muscle. During Ca2+ overload sparks serve as sites for the initiation and propagation of Ca2+ waves in myocytes. Computer simulations of spark-mediated waves are performed with model release sites that reproduce the adaptive Ca2+ release observed for the ryanodine receptor. The speed of these waves is proportional to the diffusion constant of Ca2+, D, rather than D, as is true for reaction-diffusion equations in a continuous excitable medium. A simplified "fire-diffuse-fire" model that mimics the properties of Ca2+-induced Ca2+ release (CICR) from isolated sites is used to explain this saltatory mode of wave propagation. Saltatory and continuous wave propagation can be differentiated by the temperature and Ca2+ buffer dependence of wave speed.     

Neurotransmitter modulation of small-conductance Ca2+-activated K+ channels by regulation of Ca2+ gating

Small-conductance Ca2+-activated K+ (SK) channels are widely expressed in neuronal tissues where they underlie post-spike hyperpolarizations, regulate spike-frequency adaptation, and shape synaptic responses. SK channels constitutively interact with calmodulin (CaM), which serves as Ca2+ sensor, and with protein kinase CK2 and protein phosphatase 2A, which modulate their Ca2+ gating. By recording coupled activities of Ca2+ and SK2 channels, we showed that SK2 channels can be inhibited by neurotransmitters independently of changes in the activity of the priming Ca2+ channels. This inhibition involvesSK2-associated CK2 and results from a 3-fold reduction in the Ca2+ sensitivity of channel gating. CK2phosphorylated SK2-bound CaM but not KCNQ2-bound CaM, thereby selectively regulating SK2 channels. We extended these observations to sensory neurons by showing that noradrenaline inhibits SK current and increases neuronal excitability in aCK2-dependent fashion. Hence, neurotransmitter-initiated signaling cascades can dynamically regulate Ca2+ sensitivity of SK channels and directly influence somatic excitability.     

Ca2+ sparks in skeletal muscle

The study of Ca²⁺ sparks has led to extensive new information regarding the gating of the Ca²⁺ release channels underlying these events in skeletal, cardiac and smooth muscle cells, as well as the possible roles of these local Ca²⁺ release events in muscle function. Here we review basic procedures for studying Ca²⁺sparks in skeletal muscle, primarily from frog, as well as the basic results concerning the properties of these events, their pattern and frequency of occurrence during fiber depolarization and the mechanisms underlying their termination. Finally, we also consider the contribution of different ryanodine receptor (RyR) isoforms to Ca²⁺ sparks and the number of RyR Ca²⁺ release channels that may contribute to the generation of a Ca²⁺ spark. Over the decade since their discovery, Ca²⁺ sparks have provided a wealth of information concerning the function of Ca²⁺ release channels within their intracellular environment.     

Role of phospholamban in the modulation of arterial Ca(2+) sparks and Ca(2+)-activated K(+) channels by cAMP

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR)Ca²⁺-ATPase, and this inhibition is relieved bycAMP-dependent protein kinase (PKA)-mediated phosphorylation. The roleof PLB in regulating Ca²⁺ release throughryanodine-sensitive Ca²⁺ release channels, measured asCa²⁺ sparks, was examined using smooth muscle cells ofcerebral arteries from PLB-deficient ("knockout") mice(PLB-KO). Ca²⁺ sparks were monitored opticallyusing the fluorescent Ca²⁺ indicator fluo 3 or electricallyby measuring transient large-conductance Ca²⁺-activatedK⁺ (BK) channel currents activated by Ca²⁺sparks. Basal Ca²⁺ spark and transient BK current frequencywere elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, anactivator of adenylyl cyclase, increased the frequency ofCa²⁺ sparks and transient BK currents in cerebral arteriesfrom control mice. However, forskolin had little effect on thefrequency of Ca²⁺ sparks and transient BK currents fromPLB-KO cerebral arteries. Forskolin or PLB-KO increased SRCa²⁺ load, as measured by caffeine-induced Ca²⁺transients. This study provides the first evidence that PLB is criticalfor frequency modulation of Ca²⁺ sparks and associated BKcurrents by PKA in smooth muscle.

     

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca²⁺ entry through dyhidropyridine receptor L-type Ca²⁺ channels (DHPRs) activates ryanodine receptors (RyRs) Ca²⁺-release channels, resulting in massive Ca²⁺ mobilization from the sarcoplasmic reticulum (SR). This global Ca²⁺ release arises from spatiotemporal summation of many localized elementary Ca²⁺-release events, Ca²⁺ sparks. We tested whether DHPRs modulate Ca²⁺sparks in a Ca²⁺ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca²⁺ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca²⁺ (100 nM). Under these conditions, Ca²⁺ sparks were detected with a stable frequency of 3–5 sparks·s^–1·100 µm^–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca²⁺ sparks. The DHPR modulators were also without effect on SR Ca²⁺ load (caffeine-induced Ca²⁺ transients) or sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity (Ca²⁺ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca²⁺ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca²⁺-release units in ventricular myocytes is modulated by Ca²⁺ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase; dihydropyridine receptor; sarcoplasmic reticulum     

Large currents generate cardiac Ca2+ sparks

Previous models of cardiac Ca2+ sparks have assumed that Ca2+ currents through the Ca2+ release units (CRUs) were approximately 1-2 pA, producing sparks with peak fluorescence ratio (F/F(0)) of approximately 2.0 and a full-width at half maximum (FWHM) of approximately 1 microm. Here, we present actual Ca2+ sparks with peak F/F(0) of >6 and a FWHM of approximately 2 microm, and a mathematical model of such sparks, the main feature of which is a much larger underlying Ca2+ current. Assuming infinite reaction rates and no endogenous buffers, we obtain a lower bound of approximately 11 pA needed to generate a Ca2+ spark with FWHM of 2 microm. Under realistic conditions, the CRU current must be approximately 20 pA to generate a 2- microm Ca2+)spark. For currents > or =5 pA, the computed spark amplitudes (F/F(0)) are large (approximately 6-12 depending on buffer model). We considered several factors that might produce sparks with FWHM approximately 2 microm without using large currents. Possible protein-dye interactions increased the FWHM slightly. Hypothetical Ca2+ "quarks" had little effect, as did blurring of sparks by the confocal microscope. A clusters of CRUs, each producing 10 pA simultaneously, can produce sparks with FWHM approximately 2 microm. We conclude that cardiac Ca2+ sparks are significantly larger in peak amplitude than previously thought, that such large Ca2+ sparks are consistent with the measured FWHM of approximately 2 microm, and that the underlying Ca2+ current is in the range of 10-20 pA.     

Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks

Using a combination of experimental and numerical approaches, we have tested two different approaches to calculating the sarcoplasmic reticulum (SR) Ca2+ release flux, which gives rise to cardiac muscle Ca2+ sparks. By using two-photon excited spot photolysis of DM-Nitrophen, known Ca2+ release flux time courses were generated to provide the first experimental validation of spark flux reconstruction algorithms. These artificial Ca2+ sparks show that it is possible to calculate the SR Ca2+ release waveform with reasonable accuracy, provided the flux equations reasonably reflect the properties of the experimental system. Within cardiac muscle cells, we show that Ca2+ flux reconstruction is complicated by the substantial dye binding to proteins, a factor that has not been adequately addressed in previous flux reconstruction algorithms. Furthermore, our numerical experiments suggest that the calculated time course of release flux inactivation based on conventional flux reconstruction algorithms is likely to be in error. We therefore developed novel algorithms based on an explicit dye binding scheme. When these algorithm were applied to evoked Ca2+ sparks in rat cardiac ventricular myocytes, the reconstructed Ca2+ release waveform peaked in ~5 ms and decayed with a halftime of approximately 5 ms. The peak flux magnitude was 7-12 pA, suggesting that sparks must arise from clusters of >15 ryanodine receptors.     

Modulation of Ca2+ sensitivity by cyclic nucleotides in smooth muscle from protein kinase G-deficient mice

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. We have explored cGMP- and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity.     

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.     

Regulation of the plasma membrane Ca2+ pump by cyclic nucleotides in cultured vascular smooth muscle cells

We investigated the mechanisms of Ca2+ extrusion from cultured rat aortic smooth muscle cells while monitoring changes in the cytosolic Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. 45Ca2+ efflux from these cells consisted of two major mechanisms; one was dependent on the extracellular sodium concentration (Na+o) and the other was independent of Na+o. Na+o-dependent efflux increased monotonically with increasing [Ca2+]i between 0.1 and 1.0 microM, whereas Na+o-independent efflux reached a plateau at 0.6-1 microM [Ca2+]i with a half-maximum obtained at about 0.16 microM. At [Ca2+]i below 1 microM, the latter was significantly greater than the former. Unlike the Na+o-dependent mechanism, Na+o-independent 45Ca2+ efflux was inhibited almost entirely by extracellularly added La3+ or a combination of high extracellular pH (pH 8.8) and 20 mM Mg2+. It was also inhibited, although not completely, by compound 48/80, a calmodulin antagonist, and vanadate. These results strongly suggest that Na+o-dependent and Na+o-independent 45Ca2+ effluxes occur via the Na+/Ca2+ exchanger and the ATP-dependent Ca2+ pump, respectively. Sodium nitroprusside and atrial natriuretic factor, which are agents that stimulate intracellular production of cGMP, and 8-BrcGMP significantly accelerated the Na+o-independent 45Ca2+ efflux especially at low [Ca2+]i. Forskolin, dibutyryl cAMP, and 8-Br-cAMP, however, showed no stimulation. These results suggest that the plasma membrane Ca2+ pump is regulated by cGMP but not by cAMP in intact vascular smooth muscle cells.     

Voltage dependence of Ca2+ sparks in intact cerebral arteries

Ca²⁺ sparks have beenpreviously described in isolated smooth muscle cells. Here we presentthe first measurements of local Ca²⁺ transients("Ca²⁺ sparks") in an intactsmooth muscle preparation. Ca²⁺sparks appear to result from the opening of ryanodine-sensitive Ca²⁺ release (RyR) channels in thesarcoplasmic reticulum (SR). Intracellular Ca²⁺ concentration([Ca²⁺]_i)was measured in intact cerebral arteries (40-150 µm in diameter) from rats, using the fluorescentCa²⁺ indicator fluo 3 and a laserscanning confocal microscope. Membrane potential depolarization byelevation of external K⁺ from 6 to30 mM increased Ca²⁺ sparkfrequency (4.3-fold) and amplitude (~2-fold) as well as globalarterial wall[Ca²⁺]_i(~1.7-fold). The half time of decay (~50 ms) was not affected bymembrane potential depolarization. Ryanodine (10 µM), which inhibitsRyR channels and Ca²⁺ sparks inisolated cells, and thapsigargin (100 nM), which indirectly inhibitsRyR channels by blocking the SRCa²⁺-ATPase, completely inhibitedCa²⁺ sparks in intact cerebralarteries. Diltiazem, an inhibitor of voltage-dependentCa²⁺ channels, lowered global[Ca²⁺]_iand Ca²⁺ spark frequency andamplitude in intact cerebral arteries in a concentration-dependentmanner. The frequency of Ca²⁺sparks (<1s¹ · cell¹),even under conditions of steady depolarization, was too low tocontribute significant amounts ofCa²⁺ to globalCa²⁺ in intact arteries. Theseresults provide direct evidence that Ca²⁺ sparks exist in quiescentsmooth muscle cells in intact arteries and that changes of membranepotential that would simulate physiological changes modulate bothCa²⁺ spark frequency and amplitudein arterial smooth muscle.

     

Ca2+ sparks are initiated by Ca2+ entry in embryonic mouse skeletal muscle and decrease in frequency postnatally

"Spontaneous" Ca²⁺ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca²⁺ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca²⁺ indicator dye fluo 4 to show that the frequency of spontaneous Ca²⁺ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca²⁺ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca²⁺ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl₂ (5 mM) or nifedipine (30 µM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca²⁺ sparks may be triggered by locally elevated [Ca²⁺] due to Ca²⁺ influx via dihydropyridine receptor L-type Ca²⁺ channels in embryonic mammalian skeletal muscle. calcium; ryanodine receptor; dihydropyridine receptor; muscle development     

Ca2+ modulation of Ca2+ release-activated Ca2+ channels is responsible for the inactivation of its monovalent cation current

The Ca(2+) release-activated Ca(2+) (CRAC) channel is the most well documented of the store-operated ion channels that are widely expressed and are involved in many important biological processes. However, the regulation of the CRAC channel by intracellular or extracellular messengers as well as its molecular identity is largely unknown. Specifically, in the absence of extracellular divalent cations it becomes permeable to monovalent cations with a larger conductance, however this monovalent cation current inactivates rapidly by an unknown mechanism. Here we found that Ca(2+) dissociation from a site on the extracellular side of the CRAC channel is responsible for the inactivation of its Na(+) current, and Ca(2+) occupancy of this site otherwise potentiates its Ca(2+) as well as Na(+) currents. This Ca(2+)-dependent potentiation is required for the normal functioning of CRAC channels.     

The dimeric form of Ca2+-ATPase is involved in Ca2+ transport in the sarcoplasmic reticulum

To identify the functional unit of Ca(2+)-ATPase in the sarcoplasmic reticulum, we assessed Ca(2+)-transport activities occurring on sarcoplasmic reticulum membranes with different combinations of active and inactive Ca(2+)-ATPase molecules. We prepared heterodimers, consisting of a native Ca(2+)-ATPase molecule and a Ca(2+)-ATPase molecule inactivated by FITC labelling, by fusing vesicles loaded with each type of Ca(2+)-ATPase. The heterodimers exhibited neither Ca(2+) transport nor ATP hydrolysis, suggesting that Ca(2+) transport by the Ca(2+)-ATPase requires an interaction between functional Ca(2+)-ATPase monomers. This finding implies that the functional unit of the Ca(2+)-ATPase is a dimer.     

Ca2+ release induced by cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is the most potent Ca²⁺-mobilizing agent known. It has been found in many different cell types, where it is synthesized from its precursor NAD⁺ by ADP-ribosyl cyclases. cADPR binds to Ca²⁺ channels in the endoplasmic reticulum membrane to activate a Ca²⁺-release mechanism. This release is itself potentiated by elevated cytoplasmic Ca²⁺ concentrations. Thus, cADPR may function as an endogenous regulator of Ca²⁺-induced Ca²⁺ release, and there is excitement that it may also function as a Ca²⁺-mobilizing second messenger.     

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.