Calmodulin in adult mammalian skeletal muscle: localization and effect on sarcoplasmic reticulum Ca2+ release

Calmodulin is a ubiquitous Ca2+ binding protein that binds to ryanodine rectors (RyR) and is thought to modulate its activity. Here we evaluated the effects of recombinant calmodulin on the rate of occurrence and spatial properties of Ca2+ sparks as an assay of activation in saponin-permeabilized mouse myofibers. Control myofibers exhibited a time-dependent increase and subsequent decrease in spark frequency. Recombinant wild-type calmodulin prevented the time-dependent appearance of Ca2+ sparks and decreased the derived Ca2+ flux from the sarcoplasmic reticulum during a spark by approximately 37%. A recombinant Ca2+-insensitive form of calmodulin resulted in an instantaneous increase in spark frequency as well as an increase in the derived Ca2+ flux by approximately 24%. Endogenous calmodulin was found to primarily localize to the Z-line. Surprisingly, removal of endogenous calmodulin did not alter the time dependence of Ca2+ spark appearance. These results indicate that calmodulin may not be essential for RyR1-dependent Ca2+ release in adult mammalian skeletal muscle.     

A calmodulin binding domain of RyR increases activation of spontaneous Ca2+ sparks in frog skeletal muscle

The calmodulin C lobe binding region (residues 3614-3643) on the sarcoplasmic reticulum Ca2+ release channel (RyR1) is thought to be a region of contact between subunits within RyR1 homotetramer Ca2+ release channels. To determine whether the 3614-3643 region is a regulatory site/interaction domain within RyR in muscle fibers, we have investigated the effect of a synthetic peptide corresponding to this region (R3614-3643) on Ca2+ sparks in frog skeletal muscle fibers. R3614-3643 (0.2-3.0 microM) promoted the occurrence of Ca2+ sparks in a highly cooperative dose-dependent manner, with a half-maximal activation at 0.47 microM and a maximal increase in frequency of approximately 5-fold. A peptide with a single amino acid substitution within R3614-3643 (L3624D) retained the ability to bind Ca(2+)-free calmodulin but did not increase Ca2+ spark frequency, suggesting that R3614-3643 does not modulate Ca2+ sparks by removal of endogenous calmodulin. Our data support a model in which the calmodulin binding domain of RyR1 modulates channel activity by at least two mechanisms: direct binding of calmodulin as well as interactions with other regions of RyR.     

Suramin interacts with the calmodulin binding site on the ryanodine receptor,RYR1

Apocalmodulin and Ca(2+) calmodulin bind to overlapping sites on the ryanodine receptor skeletal form, RYR1, but have opposite functional effects on channel activity. Suramin, a polysulfonated napthylurea, displaces both forms of calmodulin, leading to an inhibition of activity at low Ca(2+) and an enhancement of activity at high Ca(2+). Calmodulin binding motifs on RYR1 are also able to directly interact with the carboxy-terminal tail of the transverse tubule dihydropyridine receptor (DHPR) (Sencer, S., Papineni, R. V., Halling, D. B., Pate, P., Krol, J., Zhang, J. Z., and Hamilton, S. L. (2001) J. Biol. Chem. 276, 38237-38241). Suramin binds directly to a peptide that corresponds to the calmodulin binding site of RYR1 (amino acids 3609-3643) and blocks the interaction of this peptide with both calmodulin and the carboxyl-terminal tail of the DHPR alpha(1)-subunit. Suramin, added to the internal solution of voltage-clamped skeletal myotubes, produces a concentration-dependent increase in the maximal magnitude of voltage-gated Ca(2+) transients without significantly altering L-channel Ca(2+) channel conducting activity. Together, these results suggest that an interaction between the carboxyl-terminal tail of the DHPR alpha(1)-subunit with the calmodulin binding region of RYR1 serves to limit sarcoplasmic reticulum Ca(2+) release during excitation-contraction coupling and that suramin-induced potentiation of voltage-gated Ca(2+) release involves a relief of this inhibitory interaction.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Effects of imperatoxin A on local sarcoplasmic reticulum Ca(2+) release in frog skeletal muscle

We have investigated the effects of imperatoxin A (IpTx(a)) on local calcium release events in permeabilized frog skeletal muscle fibers, using laser scanning confocal microscopy in linescan mode. IpTx(a) induced the appearance of Ca(2+) release events from the sarcoplasmic reticulum that are approximately 2 s and have a smaller amplitude (31 +/- 2%) than the "Ca(2+) sparks" normally seen in the absence of toxin. The frequency of occurrence of long-duration imperatoxin-induced Ca(2+) release events increased in proportion to IpTx(a) concentrations ranging from 10 nM to 50 nM. The mean duration of imperatoxin-induced events in muscle fibers was independent of toxin concentration and agreed closely with the channel open time in experiments on isolated frog ryanodine receptors (RyRs) reconstituted in planar lipid bilayer, where IpTx(a) induced opening of single Ca(2+) release channels to prolonged subconductance states. These results suggest involvement of a single molecule of IpTx(a) in the activation of a single Ca(2+) release channel to produce a long-duration event. Assuming the ratio of full conductance to subconductance to be the same in the fibers as in bilayer, the amplitude of a spark relative to the long event indicates involvement of at most four RyR Ca(2+) release channels in the production of short-duration Ca(2+) sparks.     

Uncontrolled calcium sparks act as a dystrophic signal for mammalian skeletal muscle

Most excitable cells maintain tight control of intracellular Ca(2+) through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca(2+) release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca(2+) sparks in intact mammalian skeletal muscle. Spontaneous Ca(2+) sparks can be reversibly induced by osmotic shock, and participate in a normal physiological response to exercise. In dystrophic muscle with fragile membrane integrity, stress-induced Ca(2+) sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca(2+) spark response. Thus, membrane-deformation-induced Ca(2+) sparks have an important role in physiological and pathophysiological regulation of Ca(2+) signalling, and uncontrolled Ca(2+) spark activity in connection with chronic activation of store-operated Ca(2+) entry may function as a dystrophic signal in mammalian skeletal muscle.     

Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release

A Ca(2+) spark arises when a cluster of sarcoplasmic reticulum (SR) channels (ryanodine receptors or RyRs) opens to release calcium in a locally regenerative manner. Normally triggered by Ca(2+) influx across the sarcolemmal or transverse tubule membrane neighboring the cluster, the Ca(2+) spark has been shown to be the elementary Ca(2+) signaling event of excitation-contraction coupling in heart muscle. However, the question of how the Ca(2+) spark terminates remains a central, unresolved issue. Here we present a new model, "sticky cluster," of SR Ca(2+) release that simulates Ca(2+) spark behavior and enables robust Ca(2+) spark termination. Two newly documented features of RyR behavior have been incorporated in this otherwise simple model: "coupled gating" and an opening rate that depends on SR lumenal [Ca(2+)]. Using a Monte Carlo method, local Ca(2+)-induced Ca(2+) release from clusters containing between 10 and 100 RyRs is modeled. After release is triggered, Ca(2+) flux from RyRs diffuses into the cytosol and binds to intracellular buffers and the fluorescent Ca(2+) indicator fluo-3 to produce the model Ca(2+) spark. Ca(2+) sparks generated by the sticky cluster model resemble those observed experimentally, and Ca(2+) spark duration and amplitude are largely insensitive to the number of RyRs in a cluster. As expected from heart cell investigation, the spontaneous Ca(2+) spark rate in the model increases with elevated cytosolic or SR lumenal [Ca(2+)]. Furthermore, reduction of RyR coupling leads to prolonged model Ca(2+) sparks just as treatment with FK506 lengthens Ca(2+) sparks in heart cells. This new model of Ca(2+) spark behavior provides a "proof of principle" test of a new hypothesis for Ca(2+) spark termination and reproduces critical features of Ca(2+) sparks observed experimentally.     

Contribution of ryanodine receptor subtype 3 to ca2+ responses in Ca2+-overloaded cultured rat portal vein myocytes

Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca(2+) loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca(2+) responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca(2+) measurements revealed that the number of cells showing spontaneous Ca(2+) sparks was strongly increased by superfusing the vascular myocytes in 10 mm Ca(2+)-containing solution. These Ca(2+) sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca(2+) responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca(2+) responses in Ca(2+)-overloaded myocytes. Ca(2+) transients evoked by photolysis of caged Ca(2+) with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca(2+)](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca(2+) loading, the RYR3 becomes activable by caffeine and local increases in [Ca(2+)](i).     

A mathematical analysis of the generation and termination of calcium sparks

Calcium sparks are local regenerative releases of Ca(2+) from a cluster of ryanodine receptors on the sarcoplasmic reticulum. During excitation-contraction coupling in cardiac cells, Ca(2+) sparks are triggered by Ca(2+) entering the cell via the T-tubules (Ca(2+)-induced Ca(2+) release). However under conditions of calcium overload, Ca(2+) sparks can be triggered spontaneously. The exact process by which Ca(2+) sparks terminate is still an open question, although both deterministic and stochastic processes are likely to be important. In this article, asymptotic methods are used to analyze a single Ca(2+) spark model, which includes both deterministic and stochastic biophysical mechanisms. The analysis calculates both spark frequencies and spark duration distributions, and shows under what circumstances stochastic transitions are important. Additionally, a model of the coupling of the release channels via the FK-binding protein is analyzed.     

Inhibition of Ca(2+) sparks by ruthenium red in permeabilized rat ventricular myocytes

We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.     

Mechanism of calmodulin inhibition of cardiac sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor)

The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca(2+) concentrations. CaM reduced channel open probability at <10 micro M Ca(2+) by decreasing channel events and mean open times and increasing mean close times. At >10 micro M Ca(2+), CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to -50 mV and from -50 mV to +50 mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca(2+) fluxes. CaM decreased the duration of the open events after the voltage switch from -50 mV to +50 mV. In parallel experiments, a Ca(2+)-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca(2+) release.     

RYR2 proteins contribute to the formation of Ca(2+) sparks in smooth muscle

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.     

Full length ryanodine receptor subtype 3 encodes spontaneous calcium oscillations in native duodenal smooth muscle cells

Two isoforms of the ryanodine receptor subtype 3 (RYR3) have been described in smooth muscle. The RYR3 short isoform (RYR3S) negatively regulates the calcium-induced calcium release mechanism encoded by the RYR2, whereas the role of the full length isoform of RYR3 (RYR3L) was still unclear. Here, we describe RYR-dependent spontaneous Ca(2+) oscillations measured in 10% of native duodenum myocytes. We investigated the role of RYR3 isoforms in these spontaneous Ca(2+) signals. Inhibition of RYR3S expression by antisense oligonucleotides revealed that both RYR2 and RYR3L were able to propagate spontaneous Ca(2+) waves that were distinguishable by frequency analysis. When RYR3L expression was inhibited, the spontaneous Ca(2+) oscillations were never observed, indicating that RYR3S inhibited the function of RYR2. RYR2 expression inhibition led to Ca(2+) oscillations identical to those observed in control cells suggesting that RYR3S did not functionally interact with RYR3L. The presence and frequency of RYR3L-dependent Ca(2+) oscillations were dependent on sarcoplasmic reticulum Ca(2+) content as revealed by long-term changes of the extracellular Ca(2+) concentration. Our study shows that, in native duodenal myocytes, the spontaneous Ca(2+) waves are encoded by the RYR3L alone, which activity is regulated by sarcoplasmic reticulum Ca(2+) loading.     

Voltage dependence of the coupling of Ca(2+) sparks to BK(Ca) channels in urinary bladder smooth muscle

Large-conductance Ca(2+)-dependent K(+) (BK(Ca)) channels play a critical role in regulating urinary bladder smooth muscle (UBSM) excitability and contractility. Measurements of BK(Ca) currents and intracellular Ca(2+) revealed that BK(Ca) currents are activated by Ca(2+) release events (Ca(2+) sparks) from ryanodine receptors (RyRs) in the sarcoplasmic reticulum. The goals of this project were to characterize Ca(2+) sparks and BK(Ca) currents and to determine the voltage dependence of the coupling of RyRs (Ca(2+) sparks) to BK(Ca) channels in UBSM. Ca(2+) sparks in UBSM had properties similar to those described in arterial smooth muscle. Most Ca(2+) sparks caused BK(Ca) currents at all voltages tested, consistent with the BK(Ca) channels sensing approximately 10 microM Ca(2+). Membrane potential depolarization from -50 to -20 mV increased Ca(2+) spark and BK(Ca) current frequency threefold. However, membrane depolarization over this range had a differential effect on spark and current amplitude, with Ca(2+) spark amplitude increasing by only 30% and BK(Ca) current amplitude increasing 16-fold. A major component of the amplitude modulation of spark-activated BK(Ca) current was quantitatively explained by the known voltage dependence of the Ca(2+) sensitivity of BK(Ca) channels. We, therefore, propose that membrane potential, or any other agent that modulates the Ca(2+) sensitivity of BK(Ca) channels, profoundly alters the coupling strength of Ca(2+) sparks to BK(Ca) channels.     

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.     

Calcium sparks in skeletal muscle fibers

Ca(2+) sparks monitor transient local releases of Ca(2+) from the sarcoplasmic reticulum (SR) into the myoplasm. The release takes place through ryanodine receptors (RYRs), the Ca(2+)-release channels of the SR. In intact fibers from frog skeletal muscle, the temporal and spatial properties of voltage-activated Ca(2+) sparks are well simulated by a model that assumes that the Ca(2+) flux underlying a spark is 2.5 pA (units of Ca(2+) current) for 4.6 ms (18 degrees C). This flux amplitude suggests that 1-5 active RYRs participate in the generation of a typical voltage-activated spark under physiological conditions. A major goal of future experiments is to estimate this number more precisely and, if it is two or more, to investigate the communication mechanism that allows multiple RYRs to be co-activated in a rapid but self-limited fashion.     

Calcium spark properties in ventricular myocytes are altered in aged mice

This study determined whether whole cell Ca(2+) transients and unitary sarcoplasmic reticulum (SR) Ca(2+) release events are constant throughout adult life or whether Ca(2+) release is altered in aging ventricular myocytes. Myocytes were isolated from young adult (approximately 5 mo old) and aged (approximately 24 mo old) mice. Spontaneous Ca(2+) sparks and Ca(2+) transients initiated by field stimulation were detected with fluo-4. All experiments were conducted at 37 degrees C. Ca(2+) transient amplitudes were reduced, and Ca(2+) transient rise times were abbreviated in aged cells stimulated at 8 Hz compared with young adult myocytes. Furthermore, the incidence and frequency of spontaneous Ca(2+) sparks were markedly higher in aged myocytes compared with young adult cells. Spark amplitudes and spatial widths were similar in young adult and aged myocytes. However, spark half-rise times and half-decay times were abbreviated in aged cells compared with younger cells. Resting cytosolic Ca(2+) levels and SR Ca(2+) stores were assessed by rapid application of caffeine in fura-2-loaded cells. Neither resting Ca(2+) levels nor SR Ca(2+) content differed between young adult and aged cells. Thus increased spark frequency in aging cells was not attributable to increased SR Ca(2+) stores. Furthermore, the decrease in Ca(2+) transient amplitude was not due to a decrease in SR Ca(2+) load. These results demonstrate that alterations in fundamental SR Ca(2+) release units occur in aging ventricular myocytes and raise the possibility that alterations in Ca(2+) release may reflect age-related changes in fundamental release events rather than changes in SR Ca(2+) stores and diastolic Ca(2+) levels.     

Genetic ablation of caveolin-1 modifies Ca2+ spark coupling in murine arterial smooth muscle cells

L-type, voltage-dependent calcium (Ca(2+)) channels, ryanodine-sensitive Ca(2+) release (RyR) channels, and large-conductance Ca(2+)-activated potassium (K(Ca)) channels comprise a functional unit that regulates smooth muscle contractility. Here, we investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein, alters Ca(2+) spark to K(Ca) channel coupling and Ca(2+) spark regulation by voltage-dependent Ca(2+) channels in murine cerebral artery smooth muscle cells. Caveolae were abundant in the sarcolemma of control (cav-1(+/+)) cells but were not observed in cav-1-deficient (cav-1(-/-)) cells. Ca(2+) spark and transient K(Ca) current frequency were approximately twofold higher in cav-1(-/-) than in cav-1(+/+) cells. Although voltage-dependent Ca(2+) current density was similar in cav-1(+/+) and cav-1(-/-) cells, diltiazem and Cd(2+), voltage-dependent Ca(2+) channel blockers, reduced transient K(Ca) current frequency to approximately 55% of control in cav-1(+/+) cells but did not alter transient K(Ca) current frequency in cav-1(-/-) cells. Furthermore, although K(Ca) channel density was elevated in cav-1(-/-) cells, transient K(Ca) current amplitude was similar to that in cav-1(+/+) cells. Higher Ca(2+) spark frequency in cav-1(-/-) cells was not due to elevated intracellular Ca(2+) concentration, sarcoplasmic reticulum Ca(2+) load, or nitric oxide synthase activity. Similarly, Ca(2+) spark amplitude and spread, the percentage of Ca(2+) sparks that activated a transient K(Ca) current, the amplitude relationship between sparks and transient K(Ca) currents, and K(Ca) channel conductance and apparent Ca(2+) sensitivity were similar in cav-1(+/+) and cav-1(-/-) cells. In summary, cav-1 ablation elevates Ca(2+) spark and transient K(Ca) current frequency, attenuates the coupling relationship between voltage-dependent Ca(2+) channels and RyR channels that generate Ca(2+) sparks, and elevates K(Ca) channel density but does not alter transient K(Ca) current activation by Ca(2+) sparks. These findings indicate that cav-1 is required for physiological Ca(2+) spark and transient K(Ca) current regulation in cerebral artery smooth muscle cells.     

Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).     

Coupling of RYR1 and L-type calcium channels via calmodulin binding domains

In skeletal muscle the L-type Ca2+ channel directly controls the opening of the sarcoplasmic reticulum Ca2+ release channel (RYR1), and RYR1, in turn, prevents L-type Ca2+ channel inactivation. We demonstrate that the two proteins interact using calmodulin binding regions of both proteins. A recombinant protein representing amino acids 1393-1527 (D1393-1527) of the carboxyl-terminal tail of the skeletal muscle L-type voltage-dependent calcium channel binds Ca2+, Ca2+ calmodulin, and apocalmodulin. In the absence of calmodulin, D1393-1527 binds to both RYR1 and a peptide representing the calmodulin binding site of RYR1 (amino acids 3609-3643). In addition, biotinylated R3609-3643 peptide can be used with streptavidin beads to pull down [3H]PN200-110-labeled L-type channels from detergent-solubilized transverse tubule membranes. The binding of the L-type channel carboxyl-terminal tail to the calmodulin binding site on RYR1 may stabilize the contact between the two proteins, provide a mechanism for Ca2+ and/or calmodulin regulation of their interaction, or participate directly in functional signaling between these two proteins. A unique aspect of this study is the finding that calmodulin binding sequences can serve as specific binding motifs for proteins other than calmodulin.