Open-state substructure of inwardly rectifying potassium channels revealed by magnesium block in guinea-pig heart cells

Abstract

1. Outward single-channel currents through inwardly rectifying K+ channels of cardiac myocytes were studied in the open cell-attached configuration to clarify the mechanism of the rectification. The outward currents, which were not recorded in the cell-attached configuration, appeared after the inner surface of the patch was exposed to low-Mg2+ solution by rupturing a part of the cell membrane. 2. The single-channel current-voltage (I-V) relation was linear in the absence of Mg2+ and crossed the voltage axis near the equilibrium potential for K+ (EK). The channel conductance was 22 and 16 pS (15-16 degrees C) at external K+ concentrations of 150 and 40 mM, respectively. 3. The channel rapidly closed on stepping the membrane potential of the patch to values more positive than EK. Decay of the average current during depolarization was fitted with a single-exponential function. The time constant appeared voltage dependent, but also tended to increase slowly with time after opening the cell to the bath solution. 4. Mg2+ on the cytoplasmic side blocked the outward currents without affecting the inward currents. The half-saturation concentration of the Mg2+ block was 1.7 microM as examined by measuring the mean patch current at +70 mV. 5. In the presence of internal Mg2+ at a micromolar level (2-10 microM), the outward single-channel current fluctuated between four levels including two intermediate levels (sublevels) in addition to the fully open channel current and the zero-current levels. The I-V relations of each sublevel were equally spaced with an interval of about 7 pS. Corresponding sublevels were found spontaneously in the inward direction. 6. Occupancy at each level was estimated from reconstructed traces at various Mg2+ concentrations and voltages, and compared with the value predicted from the binomial theorem. At different probabilities for the blocked state, the distribution of the current levels showed reasonable agreement with the binomial theorem. These findings suggest that the inwardly rectifying K+ channel of cardiac cells is composed of three identical conducting subunits and each subunit is blocked by Mg2+ independently. 7. Dwell times in each substate were distributed exponentially. On the assumption of the above model, the blocking (mu) and unblocking (lambda) rates were calculated. The value of mu increased with higher Mg2+ concentrations or larger depolarizations, while lambda ranged between 50 and 90 s-1 and seemed independent of Mg2+. 8. Owing to the voltage-dependent block by Mg2+, the average current decayed exponentially on depolarization beyond EK.(ABSTRACT TRUNCATED AT 400 WORDS)