Inward rectification of the large conductance potassium channel in smooth muscle cells from rabbit pulmonary artery.

Large conductance Ca(2+)-dependent K+ channels were studied in smooth muscle cells enzymatically dissociated from rabbit pulmonary artery. The current-voltage relationship of single channels recorded in cell-attached patches revealed strong inward rectification, which disappeared after patch excision. Cell permeabilization with saponin, beta-escin or equinatoxin II also removed rectification. These observations imply the existence of fast open channel block by an intracellular substance(s). Application to the cytosolic side of inside-out patches of Na+ ions, mono- di- and trinucleotides, taurine, reduced and oxidized forms of glutathione, or peptides extracted from pulmonary artery smooth muscle, did not reproduce the inward rectification. Patch treatment with either alkaline phosphatase or protein kinase A alpha-subunit, which strongly affected open state probability, was also incapable of reducing the outward single channel current. Mg2+ ions applied from the cytosolic side induced concentration- and voltage-dependent block of the outward single channel currents with a Kd of 7.9 +/- 2.3 mM, resulting in inward rectification qualitatively similar to that observed in cell-attached patches. An increase in the Mg2+ concentration of the intracellular solution induced a significant decrease in the outward whole-cell current at depolarized potentials. Another putative endogenous channel blocker, the polyamine putrescine, was not effective. However, its metabolites spermidine and spermine reduced the amplitude of the outward single channel current with Kd values of 4.9 +/- 0.6 and 1.4 +/- 0.4 mM, respectively. Pre-incubation of the cells with the irreversible inhibitor of polyamine synthesis difluoromethylornithine abolished the rectification in the cell-attached patches. These results suggest that intracellular polyamines may underlie at least part of the inward rectification of the Ca2+ activated K+ channel in this tissue, but that intracellular Mg2+ is unlikely to play a major role.