Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride- sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12- 13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.     

Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites.

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca2+ (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca2+ on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low trans Ca2+ (20 microM), increasing cis Ca2+ from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating cis Ca2+ above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca2+] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca2+ enhanced the sensitivity of the channel to activating cytosolic Ca2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca2+. Potentiation of Po by increased luminal Ca2+ occurred irrespective of whether the electrochemical gradient for Ca2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca2+-sensitive site(s) at the luminal face of the channel or associated protein.     

Blockade of cardiac sarcoplasmic reticulum K+ channel by Ca2+: two-binding-site model of blockade.

Potassium countercurrent through the SR K+ channel plays an important role in Ca2+ release from the SR. To see if Ca2+ regulates the channel, we incorporated canine cardiac SR K+ channel into lipid bilayers. Calcium ions present in either the SR lumenal (trans) or cytoplasmic (cis) side blocked the cardiac SR K+ channel in a voltage-dependent manner. When Ca2+ was present on both sides, however, the block appeared to be voltage independent. A two-binding site model of blockade by an impermeant divalent cation (Ca2+) can explain this apparent contradiction. Estimates of SR Ca2+ concentration suggest that under physiological conditions the cardiac SR K+ channel is partially blocked by Ca2+ ions present in the lumen of the SR. The reduction in lumenal [Ca2+] during Ca2+ release could increase K+ conductance.     

Localization of the K+ lock-In and the Ba2+ binding sites in a voltage-gated calcium-modulated channel. Implications for survival of K+ permeability.

Using Ba2+ as a probe, we performed a detailed characterization of an external K+ binding site located in the pore of a large conductance Ca2+-activated K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba2+-blocked events. Average Ba2+ dwell time changes from 10 s at 10 mM external K+ to 100 ms in the limit of very low [K+]. Using a model where external K+ binds to a site hindering the exit of Ba2+ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-568), we calculated a dissociation constant of 2.7 mircoM for K) at this lock-in site. We also found that BK(Ca) channels enter into a long-lasting nonconductive state when the external [K+] is reduced below 4 microM using the crown ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4+ to the external solution. These results suggest that the BK(Ca) channel stability in solutions of very low [K+] is due to K+ binding to a site having a very high affinity. Occupancy of this site by K+ avoids the channel conductance collapse and the exit of Ba2+ toward the external side. External tetraethylammonium also reduced the Ba2+ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K+. It is explained in terms of a model in which the conduction pore contains Ba2+, K+, and tetraethylammonium simultaneously, with the K+ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) imply that the lock-in site and the Ba2+ sites are the external and internal ion sites of the selectivity filter, respectively.     

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel.

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.     

Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage- dependent Ca2+ binding reactions

The gating kinetics of a Ca2+-activated K+ channel from adult rat muscle plasma membrane are studied in artificial planar bilayers. Analysis of single-channel fluctuations distinguishes two Ca2+- and voltage-dependent processes: (a) short-lived channel closure (less than 1 ms) events appearing in a bursting pattern; (b) opening and closing events ranging from one to several hundred milliseconds in duration. The latter process is studied independently of the first and is denoted as the primary gating mode. At constant voltage, the mean open time of the primary gating mode is a linear function of the [Ca2+], whereas the mean closed time is a linear function of the reciprocal [Ca2+]. In the limits of zero and infinite [Ca2+], the mean open and the mean closed times are, respectively, independent of voltage. These results are predicted by a kinetic scheme consisting of the following reaction steps: (a) binding of Ca2+ to a closed state; (b) channel opening; (c) binding of a second Ca2+ ion. In this scheme, the two Ca2+ binding reactions are voltage dependent, whereas the open-closed transition is voltage independent. The kinetic constant derived for this scheme gives an accurate theoretical fit to the observed equilibrium open-state probability. The results provide evidence for a novel regulatory mechanism for the activity of an ion channel: modulation by voltage of the binding of an agonist molecule, in this case, Ca2+ ion.     

Actin modifies Ca2+ block of epithelial Na+ channels in planar lipid bilayers

The mechanism by which the cytoskeletal protein actin affects the conductance of amiloride-sensitive epithelial sodium channels (ENaC) was studied in planar lipid bilayers. In the presence of monomeric actin, we found a decrease in the single-channel conductance of alpha-ENaC that did not occur when the internal [Ca2+]free was buffered to <10 nM. An analysis of single-channel kinetics demonstrated that Ca2+ induced the appearance of long-lived closed intervals separating bursts of channel activity, both in the presence and in the absence of actin. In the absence of actin, the duration of these bursts and the time spent by the channel in its open, but not in its short-lived closed state, were inversely proportional to [Ca2+]. This, together with a lengthening of the interburst intervals, translated into a dose-dependent decrease in the single-channel open probability. In contrast, a [Ca2+]-dependent decrease in alpha-ENaC conductance in the presence of actin was accompanied by lengthening of the burst intervals with no significant changes in the open or closed (both short- and long-lived) times. We conclude that Ca2+ acts as a "fast-to-intermediate" blocker when monomeric actin is present, producing a subsequent attenuation of the apparent unitary conductance of the channel.     

A kinetic model of single and clustered IP3 receptors in the absence of Ca2+ feedback

Ca2+ liberation through inositol 1,4,5-trisphosphate receptor (IP3R) channels generates complex patterns of spatiotemporal cellular Ca2+ signals owing to the biphasic modulation of channel gating by Ca2+ itself. These processes have been extensively studied in Xenopus oocytes, where imaging studies have revealed local Ca2+ signals ("puffs") arising from clusters of IP3R, and patch-clamp studies on isolated oocyte nuclei have yielded extensive data on IP3R gating kinetics. To bridge these two levels of experimental data, we developed an IP3R model and applied stochastic simulation and transition matrix theory to predict the behavior of individual and clustered IP3R channels. The channel model consists of four identical, independent subunits, each of which has an IP3-binding site together with one activating and one inactivating Ca2+-binding site. The channel opens when at least three subunits undergo a conformational change to an "active" state after binding IP3 and Ca2+. The model successfully reproduces patch-clamp data; including the dependence of open probability, mean open duration, and mean closed duration on [IP3] and [Ca2+]. Notably, the biexponential distribution of open-time duration and the dependence of mean open time on [Ca2+] are explained by populations of openings involving either three or four active subunits. As a first step toward applying the single IP3R model to describe cellular responses, we then simulated measurements of puff latency after step increases of [IP3]. Assuming that stochastic opening of a single IP3R at basal cytosolic [Ca2+] and any given [IP3] has a high probability of rapidly triggering neighboring channels by calcium-induced calcium release to evoke a puff, optimal correspondence with experimental data of puff latencies after photorelease of IP3 was obtained when the cluster contained a total of 40-70 IP3Rs.     

Extracellular Ba2+ and voltage interact to gate Ca2+ channels at the plasma membrane of stomatal guard cells

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+](i)) that regulate K+ and Cl- channels for stomatal closure in higher-plant leaves. Under voltage clamp, the initial rate of increase in [Ca2+](i) in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating. To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P(o)) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state. Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage. We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.     

Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane.

Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.     

Delayed activation of large-conductance Ca2+-activated K channels in hippocampal neurons of the rat.

We applied a fast concentration jump system to produce step changes in Ca2+ concentration [( Ca2+]i) on the cytoplasmic side of the inside-out membrane patch, excised from isolated rat hippocampal pyramidal neurons, and examined the time course of the activation phase of the large-conductance K channel (the BK channel; approximately 266 pS) after a step rise in [Ca2+]i. Diffusion of Ca2+ from the electrode tip to the cytoplasmic surface of the patch was estimated to be almost completed in 10 ms. After a step increase in [Ca2+]i from 0.04 to 3.2-1,000 microM, the activation of the K channel started after a clear latency of 280-18 ms and proceeded along a sigmoidal function. This was in sharp contrast with the rapid deactivation that began without delay and that was completed within 50 ms. The latency in activation was not accounted for by the binding of Ca2+ to EGTA in unstirred layers in the patch, since this binding was reported to be slow, taking up to seconds at physiological pH. Calmodulin (1 microM) did not affect the delay, the activation rate, or the steady-state current level. The calmodulin inhibitors W-7 and W-5 caused flickering of the single-channel current. These results indicate a delayed activation of the BK channel after a step rise in [Ca2+]i, suggesting that the BK current does not contribute to the repolarization of the action potential. Calmodulin is probably not involved in the activation process of the channel.     

In situ characterization of the Ca2+ sensitivity of large conductance Ca2+-activated K+ channels: implications for their use as near-membrane Ca2+ indicators in smooth muscle cells.

The Ca2+ sensitivity of large conductance Ca2+- and voltage-activated K+ channels (BKV,Ca) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BKV,Ca channel activity was recorded without removing the channels from the cell. By combining patch clamp recording in the cell-attached configuration and microfluorometry of fura-2, we were able to correlate BKV,Ca channel activity with changes in cytoplasmic intracellular [Ca2+] ([Ca2+]i). The latter were induced by ionomycin, an electroneutral Ca2+ ionophore. At 0 mV, the Hill coefficient (nH) and the [Ca2+]i to attain half of the maximal BKV,Ca channel activity (Ca50) were 8 and 1 microM, respectively. The data suggest that this large Hill number was not a consequence of the difference between the near-membrane [Ca2+] ([Ca2+]s) and the bulk [Ca2+]i, indicated by fura-2. High Hill numbers in the activation by Ca2+ of BKV,Ca channels have been seen by different groups (e.g., filled squares in Fig. 4 of Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. Magleby. 1996. Biophys. J. 70:2640-2651). However, such high nH has always been considered a peculiarity rather than the rule. This work shows that a high Ca2+ cooperativity is the normal situation for BKV,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca50 did not display any significant variation among different channels or cells. It was also evident that BKV,Ca channel activity could decrease in elevated [Ca2+]i, either partially or completely. This work implies that the complete activation of BKV,Ca channels occurs with a smaller increment in [Ca2+]s than previously expected from in vitro characterization of the Ca2+ sensitivity of these channels. Additionally, it appears that the activity of BKV,Ca channels in situ does not strictly follow changes in near-membrane [Ca2+].     

Characterization of the effects of Mg2+ on Ca2+- and Sr2+-activated tension generation of skinned skeletal muscle fibers

Changes in [Mg2+] in a millimolar range have a significant inverse effect on the Ca2+- (or Sr2+)activated tension generation of skeletal muscle fibers. Single frog (Rana pipiens) semitendinosus muscle fibers were "skinned" (sarcolemma removed) and contracted isometrically in bathing solutions of varying [Ca2+] or [Sr2+] and [Mg2+] but a constant pH, [MgATP2-], [K+], [CP2-], [CPK], and ionic strength. Ca2+- (or Sr2+- )activated steady-state tensions were recorded for three [Mg2+]'s: 5 X 10(-5)M, 1 X 10(-3) M, and 2 X 10(-3) M; and these tensions were expressed as the percentages of maximum tension generation of the fibers for the same [Mg2+]. Maximum tension was not affected by [Mg2+] within Ca2+-activating or Sr2+-activating sets of solutions; however, the submaximum Ca2+-(or Sr2+)activated tension is strongly affected in an inverse fashion by increasing [Mg2+]. Mg2+ behaves as a competitive inhibitor of Ca2+ and also affects the degree of cooperativity in the system. At [Mg2+] = 5 X 10(-5)M the shape of tension versus [Ca2+] (or [Sr2+]) curve showed evidence of cooperativity of Ca2+ (or Sr2+) binding or activation of the contractile system. As [Mg2+] increased, the apparent affinity for Ca2+ or Sr2+ and cooperativity of the contractile system declined. The effect on cooperativity suggests that as [Mg2+] decreases a threshold for Ca2+ activation appears.     

Proton modulation of a Ca(2+)-activated K+ channel from rat skeletal muscle incorporated into planar bilayers

The effect of pH on the activation of a Ca-activated K+ [K(Ca)] channel from rat skeletal muscle incorporated into planar lipid bilayers was studied. Experiments were done at different intracellular Ca2+ and proton concentrations. Changes in pH modified channel kinetics only from the Ca-sensitive face of the channel. At constant Ca2+ concentration, intracellular acidification induced a decrease in the open probability (Po) and a shift of the channel activation curves toward the right along the voltage axis. The displacement was 23.5 mV per pH unit. This displacement was due to a change in the half saturation voltage (Vo) and not to a change in channel voltage dependence. The shifts in Vo induced by protons appeared to be independent of Ca2+ concentration. The slope of the Hill plot of the open-closed equilibrium vs. pH was close to one, suggesting that a minimum of one proton is involved in the proton-driven channel closing reaction. The change in Po with variations in pH was due to both a decrease in the mean open time (To) and an increase in the mean closed time (Tc). At constant voltage, the mean open time of the channel was a linear function of [Ca2+] and the mean closed time was a linear function of 1/[Ca2+]2. Changes in the internal pH modified the slope, but not the intercept of the linear relations To vs. [Ca2+] and Tc vs. 1/[Ca2+]2. On the basis of these results an economical kinetic model of the effect of pH on this channel is proposed. It is concluded that protons do not affect the open-closed reaction, but rather weaken Ca2+ binding to all the conformational states of the channel. Moreover, competitive models in which Ca2+ and H+ cannot bind to the same open or closed state are inconsistent with the data.     

Recording of intracellular Ca2+ from smooth muscle cells by sub-micron tip, double-barrelled CA2+-selective microelectrodes

Novel, double-barrelled Ca2+-selective microelectrodes with tip diameters of approximately 0.1 micron were constructed by using Simon's neutral Ca2+ ligand (ETH 1001). Concentric micropipettes were utilized for the first time for Ca2+-selective microelectrodes in which the Ca2+ ligand was incorporated into a protruding inner pipette, surrounded by an outer reference electrode. In addition, they were made from high resistance aluminosilicate glass tubing (Corning Code 1724). These Ca2+-selective electrodes had linear responses from pCa 3 to pCa 7 in the presence of constant [K+]. They provided on-line observation of changes in intracellular [Ca2+] and in the resting membrane potential in single smooth muscle cells isolated from toad stomach. The mean concentration of intracellular Ca2+ in resting cells was 163.6 +/- 20 nM (+/- SEM, n = 16). Doubling the intracellular Ca2+ level by exposure of cells to elevated [K+] was sufficient to cause shortening.     

Permeation in the dihydropyridine-sensitive calcium channel. Multi-ion occupancy but no anomalous mole-fraction effect between Ba2+ and Ca2+

We investigated the mechanism whereby ions cross dihydropyridine- sensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the single-channel level, we found no evidence of an anomalous mole- fraction effect like that reported previously for whole-cell currents in mixtures of Ba and Ca. With the total concentration of Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi- ion nature of the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions of models with only one binding site in the permeation pathway, single-channel conductance does not follow Michaelis-Menten kinetics as Ba activity is increased over three orders of magnitude. Two-fold variation in the Debye length of permeant ion solutions has little effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis- Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence of three sites helps explain both a continued rise in conductance as [Ba2+] is increased above 110 mM, and the high single-channel conductance (approximately 7 pS) with 1 mM [Ba2+] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence of an anomalous mole- fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism.     

Divalent cation block and competition between divalent and monovalent cations in the large-conductance K+ channel from Chara australis

The patch-clamp technique is used to investigate divalent ion block of the large-conductance K+ channel from Chara australis. Block by Ba2+, Ca2+, Mg2+, and Pt(NH3)4(2+) from the vacuolar and cytoplasmic sides is used to probe the structure of, and ion interactions within, the pore. Five divalent ion binding sites are detected. Vacuolar Ca2+ reduces channel conductance by binding to a site located 7% along the membrane potential difference (site 1, delta = 0.07; from the vacuolar side); it also causes channel closures with mean a duration of approximately 0.1-1 ms by binding at a deeper site (site 2, delta = 0.3). Ca2+ can exit from site 2 into both the vacuolar and cytoplasmic solutions. Cytoplasmic Ca2+ reduces conductance by binding at two sites (site 3, delta = -0.21; site 4, delta = -0.6; from the cytoplasmic side) and causes closures with a mean duration of 10-100 ms by binding to site 5 (delta = -0.7). The deep sites exhibit stronger ion specificity than the superficial sites. Cytoplasmic Ca2+ binds sequentially to sites 3-5 and Ca2+ at site 5 can be locked into the pore by a second Ca2+ at site 3 or 4. Ca2+ block is alleviated by increasing [K+] on the same side of the channel. Further, Ca2+ occupancy of the deep sites (2, 4, and 5) is reduced by K+, Rb+, NH4+, and Na+ on the opposite side of the pore. Their relative efficacy correlates with their relative permeability in the channel. While some Ca2+ and K+ sites compete for ions, Ca2+ and K+ can simultaneously occupy the channel. Ca2+ binding at site 1 only partially blocks channel conduction. The results suggest the presence of four K+ binding sites on the channel protein. One cytoplasmic facing site has an equilibrium affinity of 10 mM (site 6, delta = -0.3) and one vacuolar site (site 7, delta less than 0.2) has low affinity (greater than 500 mM). Divalent ion block of the Chara channel shows many similarities to that of the maxi-K channel from rat skeletal muscle.     

Variation of intracellular Ca2+ following Ca2+ current in heart. A theoretical study of ionic diffusion inside a cylindrical cell.

The variation in the concentration of a diffusing substance inside a cylindrical cell submitted to a time-dependent flux at the sarcolemmal membrane was studied theoretically. An application was derived to estimate the local modifications of intracellular free Ca2+ concentration ([CA2+]i) induced by the slow inward Ca2+ current (ICa) in frog heart. During a O mV voltage clamp depolarization, [Ca2+]i at the inner side of the membrane rises earlier and faster than [Ca2+]i at the center of the cell. The binding of intracellular Ca2+ to specific sites enhances the deviation between the two concentrations and may generate an accumulation-depletion process of Ca2+ near the membrane. However, it also decreases the overall [Ca2+]i. The relatively slow diffusion of sarcoplasmic Ca2+ does not significantly affect the kinetics of ICa through a modification in the Ca2+ gradient across the membrane.     

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.