Large conducting potassium channel reconstituted from airway smooth muscle

Abstract

Microsomal fractions were prepared from canine and bovine airway smooth muscle (ASM) by differential and gradient centrifugations. Surface membrane vesicles were characterized by binding assays and incorporated into planar lipid bilayers. Single-channel activities were recorded in symmetric or asymmetric K+ buffer systems and studied under voltage and Ca2+ clamp conditions. A large-conductance K(+)-selective channel (greater than 220 pS in 150 mM K+) displaying a high Ca2+, low Ba2+, and charybdotoxin (CTX) sensitivity was identified. Time analysis of single-channel recordings revealed a complex kinetic behavior compatible with the previous schemes proposed for Ca(2+)-activated K+ channels in a variety of biological surface membranes. We now report that the open probability of the channel at low Ca2+ concentration is enhanced on in vitro phosphorylation, which is mediated via an adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition to this characterization at the molecular level, a second series of pharmacological experiments were designed to assess the putative role of this channel in ASM strips. Our results show that 50 nM CTX, a specific inhibitor of the large conducting Ca(2+)-dependent K+ channel, prevents norepinephrine transient relaxation on carbamylcholine-precontracted ASM strips. It was also shown that CTX reversed the steady-state relaxation induced by vasoactive intestinal peptide and partially antagonized further relaxation induced by cumulative doses of this potent bronchodilatator. Thus it is proposed that the Ca(2+)-activated K+ channels have a physiological role because they are indirectly activated on stimulation of various membrane receptors via intracellular mechanisms.

Mechanisms underlying epithelium-dependent relaxation in rat bronchioles: analogy to EDHF-type relaxation in rat pulmonary arteries

“…In addition to bronchodilation induced by reduction of calcium oscillations and lowering of calcium through activation of sarcoplasmic Ca 2ϩ -ATPase as well as dephosphorylation of myosin light chain through increased myosin light chain phosphatase activation (26,41), activation of G-coupled ␤-adrenoceptors may lead to activation of smooth muscle BK Ca channels with subsequent relaxation sensitive to ChTX or IBTX in guinea pig trachea and human bronchi (9,43). However, in the present study, BK Ca channel blockade with IBTX did not change NS309 or salbutamol relaxation.…”

Mechanisms Responsible for Forskolin-Induced Relaxation of Rat Tail Artery

“…The results of several studies suggested that increases in cAMP concentration activate K Ca channels in bilayer 12 and whole-cell studies. 13 Calcitonin generelated peptide, a stimulus that increases cAMP concentration, also activates K ATP channels.…”

Ionic mechanisms and Ca2+handling in airway smooth muscle

“…Most studies show that blockade of large-conductance Ca 2+ -sensitive K + (KCa) channels (e.g. using charybdotoxin or iberiotoxin) has no effect on VR or resting tone [55][56][57], but there are exceptions [58]. In addition, STOCs are not recorded until Vm exceeds -30--40 mV in the smooth muscle cells of canine [13], guinea pig [13,15] and porcine [59,60] airways.…”