Identification and characterization of major ionic currents in isolated smooth muscle cells using the voltage-clamp technique

Abstract

Voltage-clamp experiments were carried out on freshly dissociated single vertebrate smooth muscle cells from the stomach muscularis ofBufo marinus. Conventional two-microelectrode methodology was used, thus avoiding rapid dialysis of the cytosol. Four major phases of current were identified upon voltage jumps from negative holding levels to more positive levels. The first phase of current was an initial, inward current. This current was blocked by external Mn²⁺ and was of the correct magnitude to account for the rising phase of the Ca²⁺-dependent, TTX-independent action potentials found in these cells. Following this initial, inward Ca²⁺ current, a large outward current was observed which reached its peak over a period of hundreds of milliseconds and then decayed over a period of seconds to a steady-state level. The peak outward current and the steady-state outward current constitute the second and third major currents. The peak outward current was the largest current observed, with a magnitude as large as tens of nanoamps whereas the inward current was at most about one nanoamp. The peak outward current was reduced more than tenfold in the presence of external TEA. It was also decreased or abolished when the preceding inward current was diminished or eliminated by using external Mn²⁺ or less negative holding potentials. In this way the peak outward current was identified as a Ca²⁺-activated K⁺ current whose slow decay was hypothesized to result from removal of internal Ca ions by cellular mechanisms following the initial rise in [Ca²⁺]_i resulting from the inward current. A fourth major current was an early transient outward current observed most clearly upon voltage jumps to more positive potentials when the inward current was eliminated by using less negative holding potentials or external Mn²⁺. A classical steady-state inactivation relationship as a function of membrane potential was constructed for the inward current. A substantial portion of this inactivation curve lies at potentials negative to the apparent threshold for activation of inward current, suggesting a true voltage-dependent inactivation. Although additional Ca²⁺-dependent inactivation could not be ruled out, neither could evidence for it be found.