MUSCARINIC STIMULATION OF TRACHEAL SMOOTH-MUSCLE CELLS ACTIVATES LARGE-CONDUCTANCE CA2-DEPENDENT K+ CHANNEL()

We investigated the regulation of the large-conductance Ca2+-dependent K+ (K(Ca)) channel by acetylcholine (ACh) in freshly dissociated trac heal smooth muscle cells. Channels were recorded in the cell-attached patch configuration, and cells were stimulated with ACh, muscarine, or caffeine. We identified K(Ca) channel activity based on 1) the voltag e dependence of channel opening; 2) the large unitary conductance (242 +/- 5 pS with symmetrical 135 mM K+); 3) dependence of the reversal p otential on the [K+] gradient, shifting 56 +/- 3 mV/10-fold change in extracellular [K+]; and 4) opening of channels after elevation of cyto solic free Ca2+ concentration ([Ca2+]i) Using the Ca2+ ionophore A2318 7. When cells were bathed either in a physiological saline solution or a solution containing 135 mM K+ (to clamp cell membrane potential nea r 0 mV), ACh caused contraction of cells and activation of voltage-dep endent channels. With 135 mM extracellular K+, the channels activated by ACh had a unitary conductance of 247 +/- 10 pS, and currents revers ed near the K+ equilibrium potential (-1 +/- 1 mV). The effects of ACh were reversible, blocked by atropine, and mimicked by muscarine. From these characteristics we conclude that muscarinic stimulation of cani ne tracheal smooth muscle cells leads to activation of the large-condu ctance K(Ca) channel. Because the K(Ca) channels were isolated from AC h by the patch pipette, the increased channel activity was probably me diated by a cytosolic second messenger. ACh shifted the threshold for K(Ca) channel opening to less positive membrane potentials, similar to that seen with elevation of [Ca2+]i. ACh caused K(Ca) channel opening in Ca2+-free solution and when Ca2+ channels were blocked with nifedi pine, consistent with ACh causing elevation of [Ca2+]i because of rele ase from intracellular stores. Furthermore, caffeine also caused activ ation of K(Ca) channels and inhibited subsequent effects of ACh. No ev idence was found for ACh suppressing K(Ca) channel activity. These res ults provide evidence that ACh causes release of Ca2+ from intracellul ar stores, eliciting contraction and activating K(Ca) channels. The ac tivation of K(Ca) channels may provide negative feedback to limit depo larization caused by muscarinic excitation.