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1. Membrane current generated by the Na(+)-Ca2+ exchange mechanism was recorded in single guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique and the intracellular free calcium concentration ([Ca2+]i) was monitored using the fluorescent probe Indo-1, applied intracellularly through a perfused patch pipette. The reversal potential of the exchanger (ENa, Ca) was measured from records of the 2 mM-Ni(2+)-sensitive current and used in an attempt to clamp [Ca2+]i at a level determined by the ionic compositions of the external and pipette solutions. 2. Measurements of ENa, Ca indicated that [Ca2+]i was close to that in the pipette solution when the holding potential was set at the ENa, Ca expected for a 3Na+:1Ca2+ exchanger. The measured value of ENa, Ca was more positive than the theoretical value when the membrane potential was held positive to ENa, Ca and the opposite was true when the holding potential was more negative than the expected ENa, Ca. 3. As Indo-1 diffused into the cell from the whole-cell clamp electrode, the intensities of the fluorescent signals measured at 405 and 480 nm increased with time, with no obvious saturation over a 10-45 min recording period. However, the ratio of these two signals reached a steady level within 5 min after rupture of the patch membrane, when the holding potential was set at the expected ENa, Ca of the exchanger. The intensity ratios measured using pipette solutions containing 600 and 803 nM [Ca2+] were almost equal to the ratios obtained extracellularly from internal solutions of identical compositions, but in experiments using pipette solutions having lower [Ca2+] the intensity ratios measured in myocytes were higher than those obtained extracellularly. 4. If the membrane was depolarized or hyperpolarized, the fluorescence ratio either increased or decreased, respectively. These changes in the fluorescence ratio were virtually blocked by the extracellular application of 2 mM-Ni2+. 5. When the concentration of bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the recording pipette was reduced from 30 to 1 mM, an increase in [Ca2+]i was observed during a depolarizing ramp pulse. The Ca2+ influx estimated by integrating the 2 mM-Ni(2+)-sensitive current during the pulse correlated with the increase in [Ca2+]i estimated from Indo-1 using the extracellular calibration curve, but the values of the influx determined directly from Indo-1 fluorescence were always larger than those calculated from the exchanger current.(ABSTRACT TRUNCATED AT 400 WORDS) |
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