Block of large conductance Ca(2+)-activated K+ channels in rabbit vascular myocytes by internal Mg2+ and Na+

Abstract

1. We studied the biophysical properties of single large conductance (> 200 pS in symmetrical K+ pipette and bath solutions) Ca(2+)-activated K+ (BKca) channels of rabbit portal vein and coronary arterial smooth muscle cells using the cell-attached and inside-out variants of the patch-clamp technique (at 22 degrees C). 2. The unitary conductance of BKca channels recorded in cell-attached patches with K+ concentrations in the range 5.4-140 mM was significantly lower than that predicted on the basis of the conductance measured in inside-out patches with symmetrical K+ pipette and bath solutions (140 mM) and the constant field equation. In cell-attached patches from cells bathed in depolarizing medium (140 mM) with 5.4 mM K+ in the pipette solution, BKca channels were difficult to detect on the physiological range of membrane potentials (approximately -50 mV). Unitary currents were smaller at all voltages in the range -50 to 0 mV and the i-V relationship exhibited strong inward rectification at potentials > 0 mV. These channels were unequivocally identified as BKca channels due to their sensitivity to caffeine (10 mM) and iberiotoxin (20 nM), and their non-stationary kinetic properties. 3. Exposure of the cytoplasmic side of excised patches to [Mg2+] in the range 0-15 mM produced two effects on BKca channel activity: the slope conductance and open probability were reduced and enhanced, respectively, in a concentration-dependent manner by this cation. The Mg(2+)-induced reduction in conductance exhibited weak voltage dependence. 4. Application of 20 mM Na+ to the internal face of BKca channels recorded in the inside-out configuration produced a flickery block at potentials > or = +20 mV resulting in reduced unitary current amplitudes and strong inward rectification of the i-V relationship. Exposure of inside-out patches to a combination of 20 mM Na+ and 2 mM Mg2+ further reduced unitary current amplitude to a level similar to the algebraic sum of the effect of each cation in isolation. 5. We conclude that Ca(2+)-dependent K+ channels of vascular smooth muscle cells display a lower unitary conductance when recorded under physiological conditions than that previously estimated on the basis of their behaviour in excised membrane patches. Our data indicate that the decreased permeation through BKca channels may be partly attributed to block by intracellular Mg2+ and Na+, which appear to interact with distinct binding sites along the inner side of the pore.