Activation of non-selective cation conductance by carbachol in freshly isolated bovine ciliary muscle cells

In smooth muscle cells freshly isolated from the bovine ciliary body, effects of carbachol (CCh) on the membrane potential and current were examined by the whole-cell clamp method. The resting membrane potential of the muscle cells used was -60 +/- 1 mV (n=111). Extracellular application of CCh (2 mu M) depolarized the cells to -15 +/- 5 mV (n=50) with an apparent increase in membrane conductance. Under voltage-clamp conditions, CCh (2 mu M) evoked an inward current which exhibited inward-going rectification and reversed the polarity at about 0 mV. Removal of Na+ from the external solution caused a reduction of the amplitude of the current and a shift of the reversal potential to the negative direction. CCh was able to elicit an inward current even under a condition where Ca2+ was the only cation producing an inwardly directed electrochemical gradient. The current was not affected by verapamil or by tetrodotoxin. The CCh-induced current was inhibited by antimuscarinic agents with the affinity sequence: atropine approximate to 4-DAMP much greater than pirenzepine > AF-DX116, indicating that the response is mediated by a muscarinic cholinoceptor that belongs to the M-3-subtype. Unlike the non-selective cation channel current in intestinal smooth muscles, which is activated by elevation of the intracellular Ca2+ concentration ([Ca2+](i)), the current of the ciliary muscle was inactivated when the [Ca2+](i) was increased. The conductance, which admits Ca2+, may serve as a pathway for Ca2+ entry required for contraction.