The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.     

Ca2+-dependent interaction of BAPTA with phospholipids

Starting from a comparative study of different Ca2+ chelators on the G-protein-induced inhibition of the CaV2.1 Ca channels, we demonstrate that BAPTA and DM-nitrophen are able to interact, in a Ca2+- and lipid-dependent manner, with phospholipid monolayers. Critical insertion pressure and sensitivity to charged lipids indicated that insertion in the lipid film may occur in biological membranes as those found on Xenopus oocytes. This novel property is not found for EGTA and EDTA and may participate to the unusual ability of BAPTA-related molecules to chelate Ca2+ ions in the very close vicinity of the plasma membrane, where most of the Ca2+-dependent signalling triggered by voltage-gated Ca2+ currents occurs.     

Unified mechanisms of Ca2+ regulation across the Ca2+ channel family

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.     

Regulation of cardiac L-type Ca2+ current in Na+-Ca2+ exchanger knockout mice: functional coupling of the Ca2+ channel and the Na+-Ca2+ exchanger

L-type Ca2+ current (I(Ca)) is reduced in myocytes from cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca2+ overload in the absence of NCX. However, Ca2+ channel expression is unchanged, suggesting that regulatory processes reduce I(Ca). We tested the hypothesis that an elevation in local Ca2+ reduces I(Ca) in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I(Ca) was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca2+ concentration on I(Ca), we used Ba2+ instead of Ca2+ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca2+ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba2+ current between WT and KO myocytes. Also, dialysis with the fast Ca2+ chelator BAPTA eliminated differences in both I(Ca) amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca2+-dependent inactivation of I(Ca) reduces I(Ca) amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca2+ that results from the absence of NCX activity inactivates some L-type Ca2+ channels. Modulation of subsarcolemmal Ca2+ by the Na+-Ca2+ exchanger may be an important regulator of excitation-contraction coupling.     

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.     

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.     

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.     

Molecular mechanism for divergent regulation of Cav1.2 Ca2+ channels by calmodulin and Ca2+-binding protein-1

Ca(2+)-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca(2+)-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca(2+) channels. Whereas CaM enhances inactivation of Ca(2+) currents through Ca(v)1.2 (L-type) Ca(2+) channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Ca(v)1.2 inactivation is unknown. Here, we identified molecular determinants in the alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of alpha(1)1.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca(2+)-independent binding of CaBP1 to the N-terminal domain (NT) of alpha(1)1.2, which was in contrast to Ca(2+)-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Ca(v)1.2 Ca(2+) currents, but spared Ca(2+)-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of alpha(1)1.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Ca(v)1.2.     

Submicroscopic Ca2+ diffusion mediates inhibitory coupling between individual Ca2+ channels.

Dihydropyridine-sensitive Ca2+ channels in heart demonstrate an important negative feedback property: they close, or inactivate, in response to prior Ca2+ entry. We now find that Ca2+ influx through one channel can selectively contribute to the inactivation of another adjacent channel, without a generalized elevation of bulk intracellular Ca2+ concentration. Intracellular application of the Ca2+ chelator BAPTA greatly diminishes such negative interactions within Ca2+ channel pairs. These findings demonstrate that Ca2+ currents are controlled not only by intrinsic channel properties, but also by local diffusive interactions among neighboring channels. Such inhibitory coupling among channels provides a concrete example of localized Ca2+ signaling, long proposed to exist on the basis of theoretical calculations.     

Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations

Single Ca2+ channel and whole cell currents were measured in smooth muscle cells dissociated from resistance-sized (100-microns diameter) rat cerebral arteries. We sought to quantify the magnitude of Ca2+ channel currents and activity under the putative physiological conditions of these cells: 2 mM [Ca2+]o, steady depolarizations to potentials between -50 and -20 mV, and (where possible) without extrinsic channel agonists. Single Ca2+ channel conductance was measured over a broad range of Ca2+ concentrations (0.5-80 mM). The saturating conductance ranged from 1.5 pS at 0.5 mM to 7.8 pS at 80 mM, with a value of 3.5 pS at 2 mM Ca (unitary currents of 0.18 pA at -40 mV). Both single channel and whole cell Ca2+ currents were measured during pulses and at steady holding potentials. Ca2+ channel open probability and the lower limit for the total number of channels per cell were estimated by dividing the whole-cell Ca2+ currents by the single channel current. We estimate that an average cell has at least 5,000 functional channels with open probabilities of 3.4 x 10(-4) and 2 x 10(-3) at -40 and -20 mV, respectively. An average of 1-10 (-40 mV and -20 mV, respectively) Ca2+ channels are thus open at physiological potentials, carrying approximately 0.5 pA steady Ca2+ current at -30 mV. We also observed a very slow reduction in open probability during steady test potentials when compared with peak pulse responses. This 4- 10-fold reduction in activity could not be accounted for by the channel's normal inactivation at our recording potentials between -50 and -20 mV, implying that an additional slow inactivation process may be important in regulating Ca2+ channel activity during steady depolarization.     

Transfer and tunneling of Ca2+ from sarcoplasmic reticulum to mitochondria in skeletal muscle

The role of mitochondrial Ca2+ transport in regulating intracellular Ca2+ signaling and mitochondrial enzymes involved in energy metabolism is widely recognized in many tissues. However, the ability of skeletal muscle mitochondria to sequester Ca2+ released from the sarcoplasmic reticulum (SR) during the muscle contraction-relaxation cycle is still disputed. To assess the functional cross-talk of Ca2+ between SR and mitochondria, we examined the mutual relationship connecting cytosolic and mitochondrial Ca2+ dynamics in permeabilized skeletal muscle fibers. Cytosolic and mitochondrial Ca2+ transients were recorded with digital photometry and confocal microscopy using fura-2 and mag-rhod-2, respectively. In the presence of 0.5 mM slow Ca2+ buffer (EGTA (ethylene glycolbis(2-aminoethylether)-N,N,N',N'-tetraacetic acid)), application of caffeine induced a synchronized increase in both cytosolic and mitochondrial [Ca2+]. 5 mM fast Ca2+ buffer (BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)) nearly eliminated caffeine-induced increases in [Ca2+]c but only partially decreased the amplitude of mitochondrial Ca2+ transients. Confocal imaging revealed that in EGTA, almost all mitochondria picked up Ca2+ released from the SR by caffeine, whereas only about 70% of mitochondria did so in BAPTA. Taken together, these results indicated that a subpopulation of mitochondria is in close functional and presumably structural proximity to the SR, giving rise to subcellular microdomains in which Ca2+ has preferential access to the juxtaposed organelles.     

ATP from subplasmalemmal mitochondria controls Ca2+-dependent inactivation of CRAC channels

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as I(CRAC). Ca2+ ions act as negative feedback regulators of I(CRAC), promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of I(CRAC) by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.     

Ca2+ dependence of the Ca2+-selective TRPV6 channel

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.     

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.     

Ca2+-induced activation and irreversible inactivation of chloride channels in the perfused plasmalemma of Nitellopsis obtusa

Experiments were carried out on the algal cells with removed tonoplast using both continuous intracellular perfusion and voltage clamp on plasmalemma. The transient plasmalemma current induced by depolarization disappeared upon perfusion with the Ca2+-chelating agent, EGTA, since the voltage-dependent calcium channels lost their ability to activate. Subsequent replacement of the perfusion medium containing EGTA by another with Ca2+ for clamped plasmalemma (-100 mV) induced an inward C1- current which showed both activation and inactivation. The maximal amplitude of the current at [C1-]in = 15 mmol/l (which is similar to that in native cells) was approximately twice that in electrically excited cell in vivo. The inactivation of C1 channels in the presence of internal Ca2+ was irreversible and had a time constant of 1-3 min. This supports our earlier suggestion (Lunevsky et al. 1983) that the inactivation of C1 channels in an intact cell (with a time constant of 1-3 s) is due to a decrease in Ca2+ concentration rather than to the activity of their own inactivation mechanism. The C1 channel selectivity sequence was following: C1- much greater than CH3SO-4 approximately equal to K+ much greater than SO2-4 (PK/PSO4 approximately 10). Activation of one half the channels occurs at a Ca2+ concentration of 2 X 10(-5) mol/l. Sr2+ also (though to a lesser extent) activated C1 channels but had to be present in a much more higher concentration than Ca2+. Mg2+ and Ba2+ appeared ineffective. Ca2+ activation did not, apparently, require participation of water-soluble intermediator including ATP. Thus, C1 channel functioning is controlled by Ca2+-, Sr2+-sensitive elements of the subplasmalemma cytoskeleton.     

Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.     

Heterologous expression of BI Ca2+ channels in dysgenic skeletal muscle

We have examined the ability of BI (class A) Ca2+ channels, cloned from rabbit brain, to mediate excitation-contraction (E-C) coupling in skeletal muscle. Expression plasmids carrying cDNA encoding BI channels were microinjected into the nuclei of dysgenic mouse myotubes grown in primary culture. Ionic currents and intramembrane charge movements produced by the BI channels were recorded using the whole-cell patch- clamp technique. Injected myotubes expressed high densities of ionic BI Ca2+ channel current (average 31 pA/pF) but did not display spontaneous contractions, and only very rarely displayed evoked contractions. The expressed ionic current was pharmacologically distinguished from the endogenous L-type current of dysgenic skeletal muscle (Idys) by its insensitivity to the dihydropyridine antagonist (+)-PN 200-110. Peak BI Ca2+ currents activated with a time constant (tau a) of approximately 2 ms and inactivated with a time constant (tau h) of approximately 260 ms (20-23 degrees C). The time constant of inactivation (tau h) was not increased by substituting Ba2+ for Ca2+ as charge carrier, demonstrating that BI channels expressed in dysgenic myotubes do not undergo Ca(2+)-dependent inactivation. The average maximal Ca2+ conductance (Gmax) produced by the BI channels was quite large (approximately 534 S/F). In contrast, the average maximal charge movement (Qmax) produced in the same myotubes (approximately 2.7 nC/microF) was quite small, being barely larger than Qmax in control dysgenic myotubes (approximately 2.3 nC/microF). Thus, the ratio Gmax/Qmax for the BI channels was considerably higher than previously found for cardiac or skeletal muscle L-type Ca2+ channels expressed in the same system, indicating that neuronal BI Ca2+ channels exhibit a much higher open probability than these L-type Ca2+ channels.     

Voltage-dependent changes of TRPV6-mediated Ca2+ currents

The physiological role and activation mechanism for most proteins of the transient receptor potential (TRP) family are unknown. This is also the case for the highly Ca(2+) selective transient receptor potential vanilloid type 6 (TRPV6) channel. Patch clamp experiments were performed on transiently transfected human embryonic kidney (HEK) cells to address this issue. Currents were recorded under various conditions of intracellular Ca(2+) buffering and monitored at the same voltage throughout. No TRPV6-mediated Ca(2+) entry was detected under in vivo Ca(2+) buffering conditions at a slightly negative holding potential; however, moderate depolarization resulted in current activation. Very similar results were obtained with different Ca(2+) chelators, either EGTA or BAPTA dialyzing the cell. TRPV6 channel activity showed a negative correlation with the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and was modulated by the membrane potential: Hyperpolarization decreases and depolarization increases TRPV6-mediated currents. Monovalent ions permeated TRPV6 channels in the absence of extracellular divalent cations. These currents were resistant to changes in the holding potential while the negative correlation to the [Ca(2+)](i) was conserved, indicating that the voltage-dependent current changes depend on blocking and unblocking the charge carrier Ca(2+) within the pore. In summary, these results suggest that the voltage dependence of TRPV6-mediated Ca(2+) influx is of physiological importance since it occurs at cytosolic Ca(2+) buffering and takes place within a physiologically relevant membrane potential range.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

Expression of a rapid,low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering

Summary Depolarization-activated outward currents ranging in amplitude from 100–1000 pA were studied in cultured, insulinsecreting HIT cells and mouse B-cells using the whole-cell patch clamp. Outward current was identified as a K current since it was blocked by K channel blockers and its tail current reversed nearE _K. The K currents of HIT cells dialyzed with internal solutions containing 0.1–10mm EGTA with no added calcium (Ca), or 10mm EGTA with 2mm added Ca, activated rapidly with depolarization. However, the stronger Ca buffer BAPTA (5mm; no added Ca) blocked the rapidly activating current to reveal an underlying more slowly activating K current. With intracellular EGTA, application of the Ca channel blocker cadmium mimicked the effect of intracellular BAPTA. These data suggest that the rapid K current was mediated by low-voltage threshold, Ca-activated K channels while the slower K current was mediated by high threshold delayed rectifier K channels. Mouse B-cells also had both K current components. Dialyzing these cells with either BAPTA (5mm, no added Ca) or high EGTA (10mm with 2mm Ca) blocked the rapid Ca-activated K current observed when cells were filled with 0.1 to 1mm EGTA. It is concluded that the extent of Ca-activated K current activation in either HIT or adult mouse B-cells depends on the degree of intracellular Ca buffering.