Tsuguhisa Ehara

1. A class of Ca2+-activated non-selective cation channel was identified in ventricular cells, which were dissociated from adult guinea-pig hearts using collagenase. 2. Under cell-attached conditions the patch electrode filled with a Na+-rich solution recorded no obvious single-channel current at the resting membrane potential. Subsequent superfusion of the ventricular cell with a Na+-free Tyrode solution induced an inward-going single-channel current as well as contracture of the cell. Kinetics of this channel were not affected by varying the membrane potential. 3. Single-channel currents showing a conductance similar to those observed in the cell-attached patches were recorded in isolated inside-out membrane patches when the inner side of the membrane was exposed to a free Ca2+ concentration ([Ca2+]i) higher than 0.3 microM. The slope conductance of the channel was 14.8 +/- 2.9 pS (mean +/- S.D., n = 17) at 20-25 degrees C. 4. The reversal potential examined in the inside-out patch was about 0 mV irrespective of the Na+-rich, K+-rich, Li+-rich or Cs+-rich solutions on either side of the membrane, thereby indicating that the channel was almost equally permeable to these cations. 5. The open probability of the channel was increased by raising [Ca2+]i with the maximum value of 0.93 +/- 0.17 (n = 4) at about 10 microM [Ca2+]i. The dose-response relation was fitted to the saturation kinetics with a Hill coefficient of 3.0 and a half-maximum concentration of 1.2 microM [Ca2+]i. 6. The gating kinetics were complex; both the open and closed time histograms showed at least two exponential components with time constants of 3.8 +/- 1.3 ms and 140 +/- 110 ms for open time and 1.8 +/- 1.1 ms and 14.9 +/- 5.3 ms for closed time (n = 4) at 10 microM [Ca2+]i. Reduction of [Ca2+]i resulted in both a decrease of the time constant of the slow component in the open time histogram and an increase of the two time constants of the closed time histogram. 7. Contribution of the channel to the whole-cell current was discussed based on an estimation of the channel density, presumably about 0.04 approximately 0.4/microns 2. Maximum activation of the channel would produce 7.2 approximately 72 nS of membrane conductance, which would explain the reported magnitude of the Ca2+-mediated background conductance of the single myocyte. The channel may also contribute, at least in part, to the transient inward current which develops in Ca2+-overloaded cardiac cells.

1. To identify the Na+- or Ca2+-induced current as Na+-Ca2+ exchange current and to determine the stoichiometry of the Na+-Ca2+ exchange, the reversal potential was measured in a wide range of external Na+ [( Na+]o) or Ca2+ [( Ca2+]o) concentrations. The Na+- or Ca2+-induced current was recorded in single ventricular cells enzymatically dispersed from guinea-pig hearts, using the technique of whole-cell voltage clamp combined with internal perfusion. 2. In the presence of 10-40 mM-Na+ and 55-803 nM-Ca2+ in the internal solution, an increase of [Ca2+]o from 0.1 to 0.5-20 mM or an increase of [Na+]o from 30 to 50-140 mM induced an extra current associated with an increase in membrane conductance. The reversal potential of these extra currents was determined from an intersection of the current-voltage (I-V) relations obtained in the absence and presence of a Na+-Ca2+ exchange blocker, Ni2+ (2 mM). 3. Ba2+ in the external solution failed to induce the extra current, but inhibited the background conductance having a reversal potential at around 0 mV. Thus, 1 mM-Ba2+ was added to all external solutions, so that a change in the background current was minimized during application of Ca2+ or Ni2+. 4. The relation between [Ca2+]o and amplitude of the Ca2+-induced current was examined in the presence and absence of Ni2+. Lineweaver-Burk analysis revealed that the action of Ni2+ on the extra current might be a mixed type of competitive and non-competitive inhibition. 5. During the application of Ca2+, the Ca2+-induced outward current decayed in a time-dependent manner, resulting in a shift of the I-V relations towards positive potentials. This current decay was inhibited by increasing the capacity of the internal Ca2+-buffer, using BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) or higher concentrations of EGTA. The result indicates that [Ca2+]i, at least under the cell membrane, changes due to ion fluxes through the Na+-Ca2+ exchange and that control of the ion concentrations within the cell is prerequisite for measuring the reversal potential of the Na+-Ca2+ exchange. 6. The shift of both the holding current and the I-V relations during stimulation of the exchange was suppressed, when the membrane potential was clamped at the equilibrium potential of 3Na+:1Ca2+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Ionic selectivity of an adrenaline-induced current was investigated in single guinea-pig ventricular cells by recording whole-cell currents using the patch clamp technique combined with internal perfusion. Other ionic currents and exchange currents known in ventricular cells were suppressed by appropriate inhibitors and the adrenaline-induced current was defined as a difference between currents obtained in the presence and absence of adrenaline. 2. The adrenaline-induced current was time independent and its I-V relation showed saturation of the inward current in the negative voltage range. 3. The reversal potential was approximately -20 mV with 140 mM-NaCl external solution and Cs(+)-rich internal solution containing 51 mM-Cl-. Replacing Na+ with various monovalent and divalent cations (Li+, K+, Rb+, Cs+, Ca2+, Sr2+ and Ba2+) produced no appreciable change in the reversal potential. 4. Varying the external Cl- concentration ([Cl-]o) in exchange for aspartate or benzenesulphonate greatly changed the reversal potential. The relationship between the reversal potential and log[Cl-]o indicated a slope of 59.5 or 53.6 mV per tenfold change in [Cl-]o in the presence of 51 or 102 mM-Cl- in the internal solution, respectively. 5. Anion substitutions did not appreciably affect the I-V relation before application of adrenaline, suggesting that the cell membrane had a low Cl- conductance in the control state. 6. 4.4'-Dinitrostilbene-2-2'-disulphonic acid (DNDS: 1-10 mM), a specific inhibitor of membrane chloride permeability, depressed the adrenaline-induced current without changing the reversal potential. 7. The results suggest strongly that the adrenaline-induced current is carried mainly by Cl-. However, the development of this current appears to depend also on external cations, since the magnitude of the adrenaline response varied depending on the external cations species, with no response in Tris-HCl or TEA-Cl solution. The external cations may facilitate the adrenaline response with a sequence of efficacy of Na+ greater than K+, Rb+ greater than Cs+, Li+, divalent cations.