Neutral carrier Na+- and Ca2+-selective microelectrodes for intracellular application.

Na+- and Ca2+-selective microelectrodes were made with Simon's neutral carrier ETH 227 and ETH 1001, respectively, and their properties were studied for intracellular application. The kNaK (selectivity coefficient for Na+ with respect to K+) values of the Na+-selective microelectrodes were in the range of 0.01-0.02, which is comparable to those of recessed-tip Na+-selective glass microelectrodes. The kNaMg values of the microelectrodes were approximately 0.005 so that the interference by intracellular Mg2+ levels could be negligible. The kNaCa values were approximately 2 and the Na+-selective microelectrodes were more selective to Ca2+ than Na+. This indicates that their intracellular application requires special care to handle Ca2+ interference under certain conditions. The kNaK, kNaMg, and kNaCa values did not depend significantly on the methods used for their determination or on the ion activity levels tested. The Nicolsky equation described well the microelectrode potentials in the mixed solutions of NaCl (1-100 mM) and KCl. Potential and resistance of the microelectrodes were stable for a long period and their response time was fast. The results indicate that the Na+-selective microlectrodes are suitable for measurements of intracellular Na ion activities. Ca2+-selective microelectrode potentials at Ca2+ concentrations lower than 10(-4) M changed significantly for the first 2-3 h and then became fairly stable. The rate of the potential change was dependent on the column length of the Ca2+-selective liquid filled. Potentials of the microelectrodes varied from 10-20 mV for Ca2+ between 10(-7) and 10(-6) M concentrations, which may be the cytosolic free-Ca2+ range. With the Ca2+ concentrations greater than 10(-6) M, the microelectrodes had potential changes of approximately 30 mV or greater for a tenfold change in Ca2+ concentration. The kCaK and kCaNa values were in the ranges of 10(-5)-10(-6) and 10(-4)-10(-5), respectively. The kCaMg values were approximately 10(-7). The results show that the Ca2+-selective microelectrodes can be used for measurements of cytosolic Ca ion activities.     

Involvement of the Ca2+-dependent K+ channel activity in the hyperpolarizing response induced by epidermal growth factor in mammary epithelial cells

Epidermal growth factor (EGF) induces a hyperpolarizing response of 5-20 mV amplitude in mouse mammary epithelial cells in culture. The amplitude of the hyperpolarizing response was reduced by more than 60% within several minutes after addition of blockers of voltage and/or Ca2+-dependent K+ channels such as tetraethylammonium (7 mM) or quinine (0.29 mM). Both nifedipine (0.15 mM), a blocker of the Ca2+ channel, and ruthenium red (2 mM), an inhibitor of the Ca2+-binding site, also reduced the amplitude of the hyperpolarizing response by more than 60%. The Ca2+ ionophore, A23187 (3.8 microM), induced a large hyperpolarization, which was 25-40 mV and lasted about 3 min. These data suggest that activity of the Ca2+-dependent K+ channel was involved in the EGF-induced hyperpolarizing response of the mammary epithelial cells.     

Ability of the Ca2+-selective microelectrodes to measure fast and local Ca2+ transients in nerve cells

The ability of the Ca2+-selective microelectrode to measure fast Ca2+ transients intracellularly is reviewed. In vitro, Ca microelectrodes can respond to Ca2+ injections with time to peaks as small as 40 ms. We present methods to improve the dynamic response of Ca microelectrodes and to make Ca-buffered solutions in high ionic strength. Examples of measurements of intracellular free Ca2+ [( Ca2+]i) transients in Aplysia neurons and in Limulus photoreceptors are shown. To show the validity of those measurements, simultaneous recordings of the Arsenazo III (AIII) absorbance and of the Ca-selective electrode potential were made in voltage-clamped neurons of the abdominal ganglion of Aplysia californica. Pressure injection of AIII to a concentration of 300-500 microM induced a rise in resting [Ca2+]i; injection of higher [AIII] led to buffering of [Ca2+]i transients. Both techniques responded to changes in resting [Ca2+]i in the same direction except that AIII showed an increase in absorbance in 0 [Ca2+]o. Voltage-clamp pulses transiently increased both the AIII absorbance and the Ca2+ electrode potential. Reducing or increasing the driving force for Ca2+ entry changed the magnitude of both signals in the right direction. Examples of spatial localization of [Ca2+]i increases and Ca2+ gradients within the cytoplasm were demonstrated using the Ca electrode. The use of optical techniques to measure local [Ca2+]i changes is briefly reviewed.     

Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.     

Evidence for Ca2+ control of the transducer mechanism in crayfish stretch receptor.

Recording from the dendrite membrane indicated a resting potential of --51.6 mV, which was reduced by inhibition of the Na+/K+ pump. Voltage clamp at rest revealed a small inward current between --50 and --80 mV and a larger outward current at clamp potentials of --40 to plus 30 mV. Using ramp-changes of muscle tension as stimuli a time-variant tension-induced inward current (TIC) became apparent, the amplitude of which decreased towards larger depolarizing voltages until at plus 18 mV the current reversed the direction. The time course of the conductance changes corresponds to similar phases in the generator potential. The outward current only responded to fast reductions in tension, decreasing transiently. A contribution of the active Na+/K+ pump to the hyperpolarizing potential response is suggested by the effects of K-removal or Na-substitution by Li+. In Na-free choline chloride media the generator potential and the TIC was depressed by 70-85%. Additional removal of Ca2+ abolished the TIC. In contrast, lowering the Ca2+ level in presence of Na+ decreased the membrane resistance and markedly enhanced the TIC (maximally eightfold at 10(-5) M Ca2+) while 75-150 mM Ca2+ or intracellular application of a Ca-ionophore had the reverse effect.     

Calcium activates and inactivates a photoreceptor soma potassium current.

Light-induced currents were measured with a two-microelectrode voltage clamp of type B photoreceptor somata, which had been isolated by axotomy from all synaptic interactions as well as from all membranes capable of generating impulse activity. In artificial seawater (ASW), light elicited a transient early inward current, INa+, which depended on Na+o and had a linear current-voltage relation and an extrapolated reversal potential of 30-40 mV (absolute). In 0-Na+ ASW, light elicited a transient short-latency outward current that dependent on K+o, increased exponentially with more positive voltages (greater than or equal to -40 mV), and reversed at -70 to -75 mV. This outward current was not blocked by Ca++ channel blockers (e.g., Cd++, Co++) or substitution of Ba++o, for Ca++o, but was reduced by iontophoretic injection of EGTA. In both ASW and 0-Na+ ASW, light also elicited a delayed, apparently inward current, which was associated with a decreased conductance, depended on K+o, increased exponentially with more positive voltages (greater than or equal to -40 mV), reversed at the equilibrium potential for K+ flux in elevated K+o was eliminated by substitution of Ba++o for Ca++o, and was greatly reduced by Cd++o or Co++o. Thus, light elicited an early Ca++-dependent K+ current, IC, and a prolonged decrease of IC. Iontophoretic injection of Ca++ through a third microelectrode caused prolonged reduction of both IC and the light-induced decrease of IC, but did not alter ICa++ or the current-voltage relation of IC. Ruthenium red (1 microM) in the external medium caused a prolongation of the light-induced decrease of IC. Iontophoretic injection of EGTA often eliminated the light-induced IC decrease while decreasing peak IC (during depolarizing steps to -5 or 0 mV) by less than one-half. EGTA injection, on the average, did not affect steady state IC but reduced the light-induced decrease of steady state IC to approximately one-third of its original magnitude. The prolonged IC decrease, elicited by dim light in the absence of light-induced IC or INa+, was more completely eliminated by EGTA injection. It was concluded that light, in addition to inducing a transient inward Na+ current, causes both a transient increase and a prolonged decrease of IC via elevation of Ca++i.     

Regulation of Ca2+-dependent K+ current by alphavbeta3 integrin engagement in vascular endothelium

Interactions between endothelial cells and extracellular matrix proteins are important determinants of endothelial cell signaling. Endothelial adhesion to fibronectin through alpha(v)beta(3) integrins or the engagement and aggregation of luminal alpha(v)beta(3) receptors by vitronectin triggers Ca2+ influx. However, the underlying signaling mechanisms are unknown. The electrophysiological basis of alpha(v)beta(3) integrin-mediated changes in endothelial cell Ca2+ signaling was studied using whole cell patch clamp and microfluorimetry. The resting membrane potential of bovine pulmonary artery endothelial cells averaged -60 +/- 3 mV. In the absence of intracellular Ca2+ buffering, the application of soluble vitronectin (200 microg/ml) resulted in activation of an outwardly rectifying K+ current at holding potentials from -50 to +50 mV. Neither a significant shift in reversal potential (in voltage clamp mode) nor a change in membrane potential (in current clamp mode) occurred in response to vitronectin. Vitronectin-activated current was significantly inhibited by pretreatment with the alpha(v)beta(3) integrin antibody LM609 by exchanging extracellular K+ with Cs+ or by the application of iberiotoxin, a selective inhibitor of large-conductance, Ca2+-activated K+ channels. With intracellular Ca2+ buffered by EGTA in the recording pipette, vitronectin-activated K+ current was abolished. Fura-2 microfluorimetry revealed that vitronectin induced a significant and sustained increase in intracellular Ca2+ concentration, although vitronectin-induced Ca2+ current could not be detected. This is the first report to show that an endothelial cell ion channel is regulated by integrin activation, and this K+ current likely plays a crucial role in maintaining membrane potential and a Ca2+ driving force during engagement and activation of endothelial cell alpha(v)beta(3) integrin.     

A novel concentric double-barrelled calcium-selective microelectrode for small cells

A novel concentric design of double-barrelled Ca2+-selective microelectrode, with an inner pipette tip that protrudes beyond an outer one, has recently been developed and is described. This configuration of pipettes was produced from concentric capillaries in one step using a horizontal pipette puller. For the tip of the inner barrel to protrude, Corning 1724 aluminosilicate glass was selected, as it has a higher melting point than the 1723 glass which is used for the outer barrel. To reduce electrode resistance the inner capillary was best made with a triangular shape. It was preferentially silanized in a dry box by injection of methyltrichlorosilane into only the inner barrel. The Ca2+ neutral carrier-based liquid membrane (ETH 1001) was back-filled from the tip to the shank of the inner pipette and above this CaCl2 solution was added. KCl, which contained EGTA and was buffered to pCa 7, was used to fill the reference barrel. These Ca2+ electrodes showed linear response with slope approximately equal to 30 mV for changes in Ca2+ concentration between 10(-3) and 10(-7) M in the presence of constant [K+]. They offer a number of advantages including a low noise level achieved by the presence of the external concentric KCl electrode, and a simple mechanical structure that allows applications to a variety of small cells.     

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.     

Two types of Ca2+ currents with different sensitivities to organic Ca2+ channel antagonists in guinea pig pancreatic alpha 2 cells

The possibility that guinea pig pancreatic alpha 2 cells are equipped with more than one type of Ca2+ channel was explored using the patch-electrode voltage-clamp technique. At a holding potential of -100 mV, a slowly developing (tau m approximately 5 ms at -40 mV assuming m2 kinetics) Ca2+ current appeared. This conductance first became detectable at potentials of about -60 mV and reached a maximum amplitude of 50-100 pA between -30 and -20 mV. During long depolarizations, it inactivated completely (tau h approximately 100 ms at -40 mV). Half-maximal steady state inactivation was observed at about -60 mV. A second, more rapidly developing (tau m approximately 2 ms at 0 mV) Ca2+ current was observed during pulses to -40 mV and above. It had a peak amplitude of 150-200 pA between 0 and 10 mV, was less dependent on the holding potential, and inactivated very little, even during long pulses. Both conductances were blocked by Co2+ but were unaffected by tetrodotoxin. The rapidly developing current differed from the slowly developing one in being sensitive to the antagonists D-600 and nifedipine, conducting Ba2+ better than Ca2+, increasing upon exposure to forskolin, and showing time-dependent decay (rundown). These findings indicate that the alpha 2 cells are equipped with two kinds of Ca2+ channels.     

Mytilus inhibitory peptide (MIP) induces a Na+-activated K+-current in snail neurons

Two microelectrode voltage-clamp and single-channel recordings were performed on D-cluster neurons of snail right parietal ganglion in order to study the properties of MIP-activated potassium current. It was found that the octapeptide member of the MIP-family, ASHIPRFVa elicits an outward current, which possesses all the properties characteristic for the hexapeptide(s) inward membrane response. The main component of the peptide elicited response is highly [K+]o dependent, however the response was attenuated in Na-free extracellular saline. The peptide elicited response was mimicked by raising the [Na+]i by pressure injection of Na+ into the cell. Single channel recordings indicated that MIP-induced outward K-current is Na-dependent. The probability to find a channel in open state increases with increasing intracellular Na+-concentration. Excised inside-out patches obtained from D-neurons contained I(K(Na)) channels could be activated by exposure of the cytoplasmic face of the patch membrane to 40 mM Na+, and 40 mM Li+, as well. The single channel current amplitude at -60 mV is 15 pA and the single channel conductance is 212 pS between -80 and 0 mV. It was concluded that MIP's activate a novel type of K+-current in the snail neurons. This current is the Na-activated K+-current. The single channel properties of the MIP activated channel is in concert with I(K(Na)) data obtained on different vertebrate and invertebrate preparations.     

Voltage-activation of high-conductance K+ channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca2+ concentration

Patch-clamp single-channel current recording experiments have been carried out on intact insulin-secreting RINm5F cells. Voltage-activation of high-conductance K+ channels were studied by selectively depolarizing the electrically isolated patch membrane under conditions with normal Ca2+ concentration in the bath solution but with or without Ca2+ in the patch pipette solution. When Ca2+ was present in the pipette, 40 mV to 120 mV depolarizing pulses (100 ms) from the normal resting potential (-70 mV) regularly evoked tetraethylammonium-sensitive large outward single-channel currents and the average open state probability during the pulses varied from about 0.015 (40 mV pulses) to 0.1 (120 mV pulses). In the absence of Ca2+ in the pipette solution the same protocol resulted in fewer and shorter K+ channel openings and the open-state probability varied from about 0.0015 (40 mV pulses) to about 0.03 (120 mV pulses). It is concluded that Ca2+ entering voltage-gated channels raises [Ca2+]i locally and thereby markedly enhances the open-state probability of tetraethylammonium-sensitive voltage-gated high-conductance K+ channels.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway.

Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.     

Simultaneous measurements of Ca2+ currents and intracellular Ca2+ concentrations in single skeletal muscle fibers of the frog

The relationship between Ca2+ current amplitudes and myoplasmic Ca2+ transients was studied in single muscle fibers. Segments of muscle fibers were voltage-clamped in a double Vaseline gap chamber. Ca2+ transients were measured as an optical signal derived from the interaction between Ca2+ and the dye antipyrylazo III. The cells were maintained at -90 mV. Ca2+ currents were detected at pulse potentials to -50 mV, reached a maximum value at 0 mV, were reduced in size for larger depolarizations, and reversed at about 40 mV. Ca2+ transients were also detected at -50 Mv and progressively increased in size with larger pulse potentials up to 10 mV. Depolarizations to voltages greater than 10 mV did not further increase the size of the transient. The magnitude and time course of transients from 10 to 70 mV were almost identical Ca2+ fluxes into the myoplasm (Ca2+ input fluxes) were calculated from the Ca2+ transients applying a removal model. The size of the input fluxes increased with depolarization up to 0 mV. Between 0 and 70 mV the peak input flux slightly increased, while the flux measured at 200 ms remained unchanged. In conclusion, Ca2+ transients and input fluxes were not reduced during pulses to large positive potentials, even though a drastic reduction of Ca2+ current occurred at these potentials. These observations make it very unlikely that a voltage-dependent Ca2+ entry is the triggering signal for contraction.     

Role of Ca2+-activated Cl- channels and MLCK in slow IJP in opossum esophageal smooth muscle

The possible contribution of Ca2+-activated Cl- channel [I(Cl(Ca))] and myosin light-chain kinase (MLCK) to nonadrenergic, noncholinergic slow inhibitory junction potentials (sIJP) was studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body and guinea pig ileum perfused with Krebs solution containing atropine (3 microM), guanethidine (3 microM), and substance P (1 microM). In opossum esophageal circular smooth muscle, resting membrane potential (MP) was -51.9 +/- 0.7 mV (n = 89) with MP fluctuations of 1-3 mV. A single square-wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.3 +/- 0.2 mV, half-amplitude duration of 635 +/- 19 ms, and rebound depolarization amplitude of 2.4 +/- 0.1 mV (n = 89). 9-Anthroic acid (A-9-C), niflumic acid (NFA), wortmannin, and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) abolished MP fluctuations, sIJP, and rebound depolarization in a concentration-dependent manner. A-9-C and NFA but not wortmannin and ML-9 hyperpolarized MP. In guinea pig ileal circular smooth muscle, nerve stimulation elicited an IJP composed of both fast (fIJP) and slow (sIJP) components, followed by rebound depolarization. NFA (200 microM) abolished sIJP and rebound depolarization but left the fIJP intact. These data suggest that in the tissues studied, activation of I(Cl(Ca)), which requires MLCK, contributes to resting MP, and that closing of I(Cl(Ca)) is responsible for sIJP.     

Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)- dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.     

Effects of Mg2+ and Ca2+ on photoinduced Euglena flagellar responses

The flagellar frequency and waveform of Euglena were analyzed under full illumination (420-700 nm) and in a restricted wavelength band (530- 700 nm) when the cells were in a medium containing Mg2+ or had been microinjected with Mg2+, Mn2+, or Ca2+ in solution. Magnesium abolished the change in flagellar frequency and the reversal in waveform that cells exhibit when illuminated by a 530-700 nm wavelength band. Under this restricted illumination, Ca2+ caused an increase in flagellar waveform reversal and a decrease in beating frequency. The flagellar motility of cells impaled on a microelectrode was examined in cells illuminated with various wavelengths.     

Effects of injecting calcium-buffer solution on [Ca2+]i in voltage-clamped snail neurons.

We have investigated why fura-2 and Ca(2+)-sensitive microelectrodes report different values for the intracellular free calcium ion concentration ([Ca(2+)]i or its negative log, pCa(i)) of snail neurons voltage-clamped to -50 or -60 mV. Both techniques were initially calibrated in vitro, using calcium calibration solutions that had ionic concentrations similar to those of snail neuron cytoplasm. Pressure injections of the same solutions at resting and elevated [Ca(2+)]i were used to calibrate both methods in vivo. In fura-2-loaded cells these pressure injections generated changes in [Ca(2+)]i that agreed well with those expected from the in vitro calibration. Thus, using fura-2 calibrated in vitro, the average resting [Ca(2+)]i was found to be 38 nM (pCa(i) 7.42 +/- 0.05). With Ca(2+)-sensitive microelectrodes, the first injection of calibration solutions always caused a negative shift in the recorded microelectrode potential, as if the injection lowered [Ca2+]i. No such effects were seen on the fura-2 ratio. When calibrated in vivo the Ca(2+)-sensitive microelectrode gave an average resting [Ca2+]i of approximately 25 nM (pCa(i) 7.6 +/- 0.1), much lower than when calibrated in vitro. We conclude that [Ca(2+)]i in snail neurons is approximately 40 nM and that Ca(2+)-sensitive microelectrodes usually cause a leak at the point of insertion. The effects of the leak were minimized by injection of a mobile calcium buffer.