In this report, we describe a novel concept of extramembrane control of channel peptide assembly and the eventual channel current modulation. Alamethicin is a peptide antibiotic, which usually forms ion channels in various association states. By introducing an extramembrane leucine zipper segment (Alm-LeuZ), the association number of alamethicin was effectively controlled to produce a single predominant channel open state. The assembly was estimated to be a tetramer, by comparison of the channel conductance with that of the template-assembled Alm-LeuZ tetramer, which was prepared by the conjugation of a maleimide-functionalized peptide template with cysteine-derivatized Alm-LeuZ segments. Employment of an extramembrane segment of a random conformation provided higher levels of channel conductance. The result exemplified the possibility of channel current control by a conformational switch of the extramembrane segments.

Upon stimulation with phenylephrine, the rabbit mesenteric artery displays endothelium-dependent and endothelium-independent rhythmic contractions in the absence and the presence of ryanodine, respectively. For examination of the involvement of the sarcoplasmic reticulum (SR) in these two types of rhythmic contractions, the mesenteric ring was suspended in an organ chamber for isometric tension recordings. Phenylephrine induced endothelium-dependent rhythmic contractions (EDRC), which were converted to endothelium-independent rhythmic contractions (EIRC) by the subsequent addition of ryanodine. Cyclopiazonic acid (CPA) also induced EIRC in the artery contracted with phenylephrine. The nifedipine-treated artery displayed neither EDRC upon phenylephrine stimulation nor EIRC by the addition of ryanodine or CPA: however, these agents relaxed the arteries. Phenylephrine induced EDRC in the artery treated with the K+ channel antagonist sparteine, but these rhythmic contractions were converted to a sustained contraction by ryanodine and CPA without producing relaxation of the artery. Ryanodine and CPA inhibited both phenylephrine-induced Ca2+ release from the SR and Ca2+ sequestration, without affecting Ca2+ influx across the plasmalemma, evaluated by monitoring agonist-induced contractions. These findings indicate that: (1) the EDRC may be attributed to Ca2+ release from the SR, which may be charged by Ca2+ influx via the voltage-dependent Ca2+ channel; and (2) the EIRC may arise from functional impairment of the SR and by the subsequent increase in the K+ efflux, presumably via the Ca(2+)-activated K+ channel.

Phenylephrine induces endothelium-independent rhythmic contractions in ryanodine-treated rabbit mesenteric arteries. To elucidate the ionic mechanism of this rhythmic behaviour, rabbit mesenteric arterial rings were suspended in an organ chamber for isometric tension studies. Yohimbine, propranolol, and atropine had no effect on these contractions, minimizing the possibility that transmitter release from nerve terminals was involved. Additionally, the oscillatory contractions were not altered by diphenhydramine, cimetidine, and indomethacin, thus ruling out the involvement of histamine and prostaglandins. This oscillatory response was completely abolished after the removal of extracellular Ca2+, as well as after Ca2+ channel blockade by diltiazem or nifedipine. Sparteine and quinidine, Ca(2+)-activated K+ channel blockade by diltiazem or nifedipine. Sparteine and quinidine, Ca(2+)-activated K+ channel antagonists, also abolished the oscillation. In contrast, tetraethylammonium and 3,4-diaminopyridine, voltage-dependent K+ channel antagonists, augmented the response. Glibenclamide, an antagonist of the ATP-sensitive K+ channel, had no effect on the rhythmic contractions. These results suggest that the rhythmic contractions observed in rabbit mesenteric arteries after ryanodine treatment were caused by the movement of Ca2+ and K+ across the plasmalemma via the voltage-dependent Ca2+ channel and the Ca(2+)-activated K+ channel, respectively.