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

Abstract

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.

Characterization of the large-conductance Ca-activated K channel in myocytes of rat saphenous artery

“…When single BK channel recordings were taken from intact myocytes, we found that the value of the outward current was significantly less than that recorded from excised patches, a finding in line with previous reports [28,44]. In particular, the current was markedly reduced at high voltages (presumably due to the high voltage-dependent block of intracellular polyamines, and Mg and Na ions [11,28,44]), whereas only minor inhibition (smaller than 30%) of the current was observed at physiologically relevant membrane potentials.…”

“…In cell-attached patches BK channel conductance was smaller than that recorded following excision of the patch. This observation has been already documented for BK channels in other smooth muscle tissues [28,44], and interpreted to be due to voltage-dependent block of the BK channel by intracellular Mg and Na ions, and polyamines. This behaviour is illustrated in Fig.…”

BK Channels – Focus on Polyamines, Ethanol/Acetaldehyde and Hydrogen Sulfide (H2S)

“…BK channels of rabbit pulmonary smooth muscle in contrast to other cells exhibit strong rectification (Snetkov et al, 1996). This was attributed to the presence of spermine and spermidine but not putrescine in the cytoplasm.…”

Equinatoxins: A Review

“…In smooth muscle cells from rabbit pulmonary artery EqT II and other permeabilizing agents caused removal of inward rectification in large conductance Ca 2+ -activated potassium channels that could not be explained by pore-formation and cell permeabilization (Snetkov et al 1996). It is well established that pore formation in erythrocytes requires at least 0.1 nM, but in most other cells at least 100 nM EqT II.…”