Endogenous intracellular calcium buffering and the activation/inactivation of HVA calcium currents in rat dentate gyrus granule cells.

Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca2+ channels. The kinetics of whole-cell Ca2+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca2+ buffers, were fit best by Hodgkin-Huxley kinetics (m2h), and were indistinguishable from those recorded with the nystatin perforated patch method. In the absence of exogenous Ca2+ buffers, inactivation of HVA Ca2+ channels was a predominantly Ca(2+)-dependent process. The contribution of endogenous Ca2+ buffers to the kinetics of inactivation was investigated by comparing currents recorded from control cells to currents recorded from neurons that have lost a specific Ca(2+)-binding protein, Calbindin-D28K (CaBP), after kindling-induced epilepsy. Kindled neurons devoid of CaBP showed faster rates of both activation and inactivation. Adding an exogenous Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), to the intracellular solution largely eliminated inactivation in both control and kindled neurons. The results are consistent with the hypothesis that endogenous intraneuronal CaBP contributes significantly to submembrane Ca2+ sequestration at a concentration range and time domain that regulate Ca2+ channel inactivation.     

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine. Loading cells with low concentrations of the cell-permeant esters of the artificial Ca(2+)-buffers EGTA or BAPTA, resulted in fewer cells with a distinct "baseline" oscillatory pattern, and loading with higher concentrations of BAPTA almost completely abolished them. In HeLa-CaBP cells, stimulation with 1 microM histamine resulted in individual Ca(2+) spikes that had a flattened profile when compared to control cells; peak [Ca(2+)](i) was lowered, the rate of increase in [Ca(2+)](i) was slower and transients were prolonged. When compared to HeLa-pCINeo cells, loading with EGTA or BAPTA, or transfection of CaBP, significantly reduced the propagation velocity (by up to 60%) of Ca(2+) waves induced by exposure to 100 microM histamine. We conclude that intracellular Ca(2+) buffering exerts a significant influence on global Ca(2+) responses in HeLa cells and the propagation of Ca(2+) waves that underlie them. The relative effectiveness of different Ca(2+) buffers, including CaBP, appears to be particularly dependent upon the rapidity of their binding kinetics, with BAPTA being the most effective.     

Protein kinase activation increases insulin secretion by sensitizing the secretory machinery to Ca2+

Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca(2+), we characterize three kinetically different pools of vesicles in rat pancreatic beta-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is approximately 20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca(2+), we find that the HCSP is a distinct population of vesicles from those colocalized with Ca(2+) channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca(2+).     

Endogenous cytosolic Ca(2+) buffering is necessary for TRPM4 activity in cerebral artery smooth muscle cells

The melastatin transient receptor potential (TRP) channel, TRPM4, is a critical regulator of smooth muscle membrane potential and arterial tone. Activation of the channel is Ca(2+)-dependent, but prolonged exposures to high global Ca(2+) causes rapid inactivation under conventional whole-cell patch clamp conditions. Using amphotericin B perforated whole cell patch clamp electrophysiology, which minimally disrupts cytosolic Ca(2+) dynamics, we recently showed that Ca(2+) released from 1,2,5-triphosphate receptors (IP(3)R) on the sarcoplasmic reticulum (SR) activates TRPM4 channels, producing sustained transient inward cation currents (TICCs). Thus, Ca(2+)-dependent inactivation of TRPM4 may not be inherent to the channel itself but rather is a result of the recording conditions. We hypothesized that under conventional whole-cell configurations, loss of intrinsic cytosolic Ca(2+) buffering following cell dialysis contributes to inactivation of TRPM4 channels. With the inclusion of the Ca(2+) buffers ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 10mM) or bis-ethane-N,N,N',N'-tetraacetic acid (BAPTA, 0.1mM) in the pipette solution, we mimic endogenous Ca(2+) buffering and record novel, sustained whole-cell TICC activity from freshly-isolated cerebral artery myocytes. Biophysical properties of TICCs recorded under perforated and whole-cell patch clamp were nearly identical. Furthermore, whole-cell TICC activity was reduced by the selective TRPM4 inhibitor, 9-phenanthrol, and by siRNA-mediated knockdown of TRPM4. When a higher concentration (10mM) of BAPTA was included in the pipette solution, TICC activity was disrupted, suggesting that TRPM4 channels on the plasma membrane and IP(3)R on the SR are closely opposed but not physically coupled, and that endogenous Ca(2+) buffer proteins play a critical role in maintaining TRPM4 channel activity in native cerebral artery smooth muscle cells.     

Ca(2+) -independent effects of BAPTA and EGTA on single-channel Cl(-) currents in brown adipocytes

The Cl(-) channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl(-) channels (ORCC). To study tentative Ca(2+) regulation of these channels, we attempted to control Ca(2+) levels at the cytoplasmic side of the inside-out membrane patches with Ca(2+)-chelating agents. However, we found that the commonly used Ca(2+)-chelators EGTA and BAPTA by themselves influenced the Cl(-) channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca(2+)-chelators (acting from the cytoplasmic side) on the single Cl(-) channel currents in patch-clamp experiments. Using fixed (1-2 mM) concentrations of chelators, two types of Cl(-) channels were identified, as discriminated by their reaction to the Ca(2+)-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca(2+). The anionic classical Cl(-) channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca(2+) level control in themselves could fundamentally affect the behaviour of Cl(-) channels.     

Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media.

Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.     

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.     

Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.     

Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.     

Cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in hela cells

Results of recent studies using BAPTA/AM have raised a serious question on whether Ca(2+) signal is truly involved in regulating the progression of apoptosis. To resolve this question, we examined the differential effects of three different Ca(2+) signaling blockers (BAPTA/AM, membrane-impermeant BAPTA, and heparin) on UV-induced apoptosis in HeLa cells. We found that although the membrane-permeable form of BAPTA (i.e., BAPTA/AM) could not inhibit cell death, the membrane-impermeant form of BAPTA, loaded into the cytosol by electroporation, clearly protected cells from entering apoptosis. Furthermore, when we injected heparin to block Ca(2+) release from the endoplasmic reticulum (ER) to cytosol, apoptosis was greatly suppressed. These findings strongly suggest that elevation of cytosolic Ca(2+) is part of the signal that drives the progression of apoptosis. The negative result of BAPTA/AM is probably due to its dual effect on subcellular Ca(2+) distribution; besides suppressing the Ca(2+) elevation in cytosol, BAPTA/AM can also enter into the ER to reduce the free Ca(2+) level there. The depletion of Ca(2+) in ER is believed to stimulate apoptosis and thus would counterbalance the protection effect of BAPTA/AM in suppressing the cytosolic Ca(2+) elevation.     

Development and dissipation of Ca(2+) gradients in adrenal chromaffin cells

We used pulsed laser imaging to measure the development and dissipation of Ca(2+) gradients evoked by the activation of voltage-sensitive Ca(2+) channels in adrenal chromaffin cells. Ca(2+) gradients appeared rapidly (<5 ms) upon membrane depolarization and dissipated over several hundred milliseconds after membrane repolarization. Dissipation occurred with an initial fast phase, as the steep gradient near the membrane collapsed, and a slower phase as the remaining shallow gradient dispersed. Inhibition of active Ca(2+) uptake by the endoplasmic reticulum (thapsigargin) and mitochondria (carbonylcyanide p-trifluoro-methoxyphenylhydrazone/oligomycin) had no effect on the size of Ca(2+) changes or the rate of gradient dissipation, suggesting that passive endogenous Ca(2+) buffers are responsible for the slow Ca(2+) redistribution. We used a radial diffusion model incorporating Ca(2+) diffusion and binding to intracellular Ca(2+) buffers to simulate Ca(2+) gradients. We included a 3D optical sectioning model, simulating the effects of out-of-focus light, to allow comparison with the measured gradients. Introduction of a high-capacity immobile Ca(2+) buffer, with a buffer capacity on the order of 1000 and appropriate affinity and kinetics, approximated the size of the Ca(2+) increases and rate of dissipation of the measured gradients. Finally, simulations without exogenous buffer suggest that the Ca(2+) signal due to Ca(2+) channel activation is restricted by the endogenous buffer to a space less than 1 microm from the cell membrane.     

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.     

Excitation-induced Ca(2+) influx in rat soleus and EDL muscle: mechanisms and effects on cellular integrity

In rat skeletal muscle, electrical stimulation increases Ca(2+) influx leading to progressive accumulation of calcium. Excitation-induced Ca(2+) influx in extensor digitorum longus (EDL; fast-twitch fibers) and soleus muscle (slow-twitch fibers) is compared. In EDL and soleus, stimulation at 40 Hz increased (45)Ca uptake 34- and 21-fold and (22)Na uptake 17- and 7-fold, respectively. These differences may be related to the measured 70% higher concentration of Na(+) channels in EDL. Repeated stimulation at 40 Hz elicited a delayed release of lactic acid dehydrogenase (LDH) from EDL (11-fold increase) and soleus (5-fold increase). Continuous stimulation at 1 Hz increased LDH release only from EDL (18-fold). This was associated with increased Ca(2+) content and was augmented at high extracellular Ca(2+) concentration ([Ca(2+)](o)) and suppressed at low [Ca(2+)](o). The data support the hypothesis that excitation-induced Ca(2+) influx is mediated in part by Na(+) channels and that the ensuing increase in intracellular Ca(2+) induces cellular damage. This is most pronounced in EDL, which may account for the repeated observation that prolonged exercise leads to preferential damage to fast-twitch fibers.     

Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog.

Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]i) were monitored optically in hair cells mechanically isolated from frog semicircular canals using the membrane-impermeant form of the Ca(2+)-selective dye Oregon Green 488 BAPTA-1 (OG, 100 microM). Cells stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca(2+) entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the cell body. [Ca(2+)]i at individual hotspots rose with a time constant tau1 approximately 70 ms and decayed with a bi-exponential time-course (tau2 approximately 160, tau3 approximately 2500 ms) following a 160 ms depolarization to -20 mV. With repeated stimulation [Ca(2+)]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca(2+) channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca(2+)channels are distributed at different densities in distinct hotspots. No diffusion barrier other than the nuclear region was found in the cytosol, so that, during a prolonged depolarization (lasting up to 1s), Ca(2+) was able to reach the cell apical ciliated pole. The effective Ca(2+) diffusion constant, measured from the progression of Ca(2+) wavefronts in the cytosol, was approximately 57 microm(2)/s. Our results indicate that in these hair cells, buffered diffusion of Ca(2+) proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers.     

Inhibition of phospholipase C activity in Drosophila photoreceptors by 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) and di-bromo BAPTA

In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP2. In cells loaded via patch pipettes with varying concentrations of Ca2+ buffered by 4 mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca2+ (maximum at "100 nM", approximately 10-fold inhibition at <10nM or approximately 1 microM). However, experiments where the total BAPTA concentration was varied whilst free [Ca2+] was maintained constant indicated that inhibition of PLC at higher (>100 nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 approximately 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 approximately 1mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca2+ chelators, whilst non-physiologically low (<100 nM) levels of Ca2+ suppress both basal and light-induced PLC activity.     

Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels

Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.     

Effects of rapid buffers on Ca2+ diffusion and Ca2+ oscillations.

Based on realistic mechanisms of Ca2+ buffering that include both stationary and mobile buffers, we derive and investigate models of Ca2+ diffusion in the presence of rapid buffers. We obtain a single transport equation for Ca2+ that contains the effects caused by both stationary and mobile buffers. For stationary buffers alone, we obtain an expression for the effective diffusion constant of Ca2+ that depends on local Ca2+ concentrations. Mobile buffers, such as fura-2, BAPTA, or small endogenous proteins, give rise to a transport equation that is no longer strictly diffusive. Calculations are presented to show that these effects can modify greatly the manner and rate at which Ca2+ diffuses in cells, and we compare these results with recent measurements by Allbritton et al. (1992). As a prelude to work on Ca2+ waves, we use a simplified version of our model of the activation and inhibition of the IP3 receptor Ca2+ channel in the ER membrane to illustrate the way in which Ca2+ buffering can affect both the amplitude and existence of Ca2+ oscillations.     

Calmodulin modulates initiation but not termination of spontaneous Ca2+ sparks in frog skeletal muscle

Calmodulin is a ubiquitous Ca(2+) sensing protein that binds to and modulates the sarcoplasmic reticulum Ca(2+) release channel, ryanodine receptor (RYR). Here we assessed the effects of calmodulin on the local Ca(2+) release properties of RYR in permeabilized frog skeletal muscle fibers. Fluorescently labeled recombinant calmodulin in the internal solution localized at the Z-line/triad region. Calmodulin (0.05-5.0 micro M) in the internal solution (free [Ca(2+)](i) approximately 50-100 nM) initiated a highly cooperative dose-dependent increase in Ca(2+) spark frequency, with a half-maximal activation (K) of 1.1 micro M, a Hill coefficient (n) of 4.2 and a fractional maximal increase in frequency (R) of 17-fold. A non-Ca(2+) binding mutant of calmodulin elicited a similar highly cooperative dose-dependent increase in spark frequency (K = 1.0 micro M; n = 3.7; R = 12-fold). Spatiotemporal properties of Ca(2+) sparks were essentially unaffected by either wild-type or mutant calmodulin. An N-terminal extension of calmodulin, (N+3)calmodulin, that binds to but does not activate RYR at nM [Ca(2+)] in sarcoplasmic reticulum vesicles, prevented the calmodulin-induced increase in spark frequency. These data suggest that exogenous Ca(2+)-free calmodulin cooperatively sensitizes the Ca(2+) release channel to open, but that Ca(2+) binding to the added calmodulin does not play a significant role in the termination of Ca(2+) sparks.     

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells.

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.