INWARD RECTIFICATION OF THE LARGE-CONDUCTANCE POTASSIUM CHANNEL IN SMOOTH-MUSCLE CELLS FROM RABBIT PULMONARY-ARTERY

Large conductance Ca2+-dependent K+ channels were studied in smooth mu scle cells enzymatically dissociated from rabbit pulmonary artery. The current-voltage relationship of single channels recorded in cell-atta ched patches revealed strong inward rectification, which disappeared a fter patch excision. Cell permeabilization with saponin, beta-escin or equinatoxin II also removed rectification. These observations imply t he existence of fast open channel block by an intracellular substance( s). Application to the cytosolic side of inside-out patches bf Na+ ion s, mono-, di- and trinucleotides, taurine, reduced and oxidized forms of glutathione, or peptides extracted from pulmonary artery smooth mus cle, did not reproduce the inward rectification. Patch treatment with either alkaline phosphatase or protein kinase A a-subunit, which stron gly affected open state probability, was also incapable of reducing th e outward single channel current. Mg2+ ions applied from the cytosolic side induced concentration- and voltage-dependent block of the outwar d single channel currents with a K-d of 7.9 +/- 2.3 mM, resulting in i nward rectification qualitatively similar to that observed in cell-att ached patches. An increase in the Mg2+ concentration of the intracellu lar 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 me tabolites spermidine and spermine reduced the amplitude of the outward single channel current with K, values of 4.9 +/- 0.6 and 1.4 +/- 0.4 mM, respectively. Pre-incubation of the cells with the irreversible in hibitor of polyamine synthesis difluoromethylornithine abolished the r ectification in the cell-attached patches. These results suggest that intracellular polyamines may underlie at least part of the inward rect ification of the Ca2+-activated K+ channel in this tissue, but that in tracellular Mg2+ is unlikely to play a major role.