The Electrophysiological Property of the Sodium Channel in Freshly Isolated Bovine Ciliary Smooth Muscle Cells

Abstract

Abstract: : Purpose: To characterize the electrophysiological properties of activation and inactivation of the voltage–gated sodium channel recorded from the bovine ciliary muscle cells Materials and methods: Smooth muscle cells dispersed from the bovine ciliary body by collagenase treatment were used. Membrane currents were recorded by whole–cell voltage clamp patch–clamp experiment. Unless otherwise mentioned, the holding potential was set at –80 mV. The bath was perfused with a HEPES–Krebs solution (pH 7.4, 30 °C), and borosilicate–glass pipette electrodes were filled with 100 mM–Cs⁺ solution containing 70 nM–Ca²⁺ and 200 µM–GTP (pH 7.0). Results: In the isolated ciliary muscle cells, an inward current having a peak (–40 to –200 pA) at –10 ± 2 mV (n=4) was evoked by applying a ramp voltage pulse ascending from –120 to +80 mV at the rate of 1 V/s. No inward current was observed when a ramp command pulse descending from +80 to –120 mV was applied. The currents evoked by ascending voltage pulses were inhibited by extracellular application of tetrodotoxin in a dose–dependent manner (K_i=3.1 nM, n=21), whereas they were not affected by either carbachol (2 µM) or verapamil (1 µM). Essentially similar voltage dependence and sensitivity to tetrodotoxin were observed for inward currents invoked by rectangular voltage pulses (110 to 80 mV in 10 mV step, 100 ms duration). The inward currents invoked in response to the test commands to 0 mV was strongly inhibited by applying a depolarizing pre–pulse (–80 to +80 mV, 5 s duration) immediately prior to application of the test commands. The peak current amplitude was steeply reduced from the control level to zero in the pre–pulse range from –70 to –30 mV. A 50 % inactivation occurred at the pre–pulse potential of –46 ± 2 mV (n=13). When all extracellular monovalent and divalent metal cations were isosmotically replaced with Ca²⁺, small but clearly monitored inward current was observed. Conclusions: The bovine ciliary muscle possesses a voltage–dependent sodium channel similar to the ones widely distributed in neurons or skeletal muscle. This channel, which is measurably permeable to Ca²⁺, may serve as a pathway for Ca²⁺ entry from the extracellular space in the early phase of the contraction.

Keywords: ciliary muscle • ion channels