Involvement Of Anion Channel(s) In The Modulation Of The Transient Outward Potassium Channel In Rat Ventricular Myocytes

The durations of action potentials (AP) in the mammalian myocardiaum is largely determined by voltage-gated K+ channels, and changes of voltage-gated K+ channels are closely related with cardiac hypertrophy, heart failure and arrhythmogenesis. Two broad classes of voltage-gated K+ channel currents have been distinguished based on differences in time- and voltage-dependent properties and pharmacological sensitivities. One is a rapidly activating and inactivating transient outward current (I_t0). The other is a slowly activating, delayed-rectifier K+ current (I_k). I_to is mainly responsible for the initial rapid phase of the AP repolarization. A lot of studies have been done about properties of I_t0. Initial studies found that I_t0 was carried primarily by Cl- by using anion substitution and K+ channel blockers. However, later studies revealed that K+, rather than Cl^, was the main charge carrier of I_t0. At present, it has been confirmed that I_t0 has two transient outward current components, referred to as I_t01 and I_t02. I_t01 is a K+ current; I_t02 is a Ca²⁺ -activated Cl current. In rat cardiac ventricular myocytes, only I_t01 is foundand referred to as I_t0 in the present study, but still influenced by anionsubstitude. The mechanism of the action of anions on I_t0 is not clear.In the present study, we explored the mechanism of the action of anions on I_t0 by using whole-cell patch-clamp technique in single isolated rat cardiac ventricular myocytes.1. Objectives(1) To observe the action of less permeable anions, aspartate (Asp⁾, glutamate (Glu-) and gluconate (Glc-) on rat cardiac ventricular I_t0.(2) To observe the action of permeable anions, F-, Br- and NO₃- on rat cardiac ventricular I_t0.(3) To observe the action of anions on rat cardiac ventricular I_t0 by using cytoskeleton medicine.(4) To study Cl- channel(s) that is(are) probably involved in the regulation of I_t0 channel in rat cardiac ventricular myocytes.2. Methods(1) Rat ventricular myocytes were isolated by conventional enzyme action.(2) Immunofiuorescence staining experiments were done to observe the change of cytoskeleton.(3) The action of different anions on I_t0 and Cl- current were observed by using whole-cell patch-clamp technique.3. Main results(1) The rat cardiac ventricular I_t0 was sensitive to blockade by the K+ channel blocker 4-aminopyridine (4-AP) and was abolished by Cs+ substitution for intracellular K+, indicating that the current was the4-AP-sensitive and Ca2+-independent K+ current.(2) Equimolar replacement of the extracellular Cl with less permeable anions, Asp", Glu" and Glc", signifinantly suppressed the amplitude of 7t0 in Na+-rich bath solution. Perfusing the cells with Na+-free solution (replacement of extracellular Na+ with the impermeable monovalent cation NMDG+) did not prevent the inhibitory effect of Asp" substitutes. Oubabin had no significant effect on /t0.(3) /t0 was facilitated when the extracellular Cl was replaced with equimolar permeable anions, F^, Br and NO3^.(4) The facilitative effect of different anions on the activation of /t0 was in the following sequence: NO3" > Cl" > Br" > F Glc" > Glu" > Asp".(5) The effects of different anions on the steady-state inactivation curve of /t0 were different. Less permeable anions shifted the steady-state inactivation curve of /t0 to negative potentials. Permeable Br" did not significantly influence the steady-state inactivation curve of /t0 and NC>3 shifted the steady-state inactivation curve of /t0 to positive potential. The ability of various anions to shift the steady-state inactivation curve of /t0 to more positive potentials was in the following sequence: NO3 > Cl Br" > Glc" > Glu" > Asp".(6) The steady-state inactivation curve of /t0 was shifted to negative potential and the shift of the steady-state inactivation curve of/t0 by Asp" was inhibited, when cytoskeleton was destroyed by cytochalasin D (CytD).(7) Confocal images showed that cells pretreated with Asp^-rich external solution did not induce significant changes in the average...