Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na 1 Channels

Human heart Na 1 channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na 1 currents measured using 150 mM intracellular Na 1 . The kinetics of decaying outward Na 1 current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li 1 , Na 1 , K 1 , Rb 1 , and Cs 1 than for the organic cations Tris, tetramethylammonium, N -methylglucamine, and choline. In whole cell recordings, raising [Na 1 ] o from 10 to 150 mM increases the rate of recovery from slow inactivation at 2 140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na 1 ] o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at 1 60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na 1 ] o . However raising [Na 1 ] o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na 1 ] o , suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate. key words: sodium channels • gating • single channel recording • kinetics i n t r o d u c t i o n In the previous paper we reported effects of extracellular cations on the peak open probability ( P open ) 1 of cardiac Na 1 currents (Townsend et al., 1997). Low concentrations of permeant cations were associated with a reduction of P open at depolarized voltages with little effect on the kinetics of activation. The process of fast inactivation is also relatively insensitive to extracellular Na 1 concentration (Armstrong and Bezanilla, 1974; Oxford and Yeh, 1985; Correa and Bezanilla, 1994; O’Leary et al., 1994; Bezanilla and Correa, 1995; Tang et al., 1996). Fast inactivation is believed to involve a cytoplasmically-located gate (Rojas and Armstrong, 1971; Stühmer et al., 1989; Moorman et al., 1990; Patton et al., 1992; West et al., 1992). Another type of inactivation with much slower kinetics (seconds versus milliseconds) co-exists with fast inactivation in all Na 1 channels (Adelman and Palti, 1969; Ruff et al., 1987; Simoncini and Stühmer, 1987; Ruben et al., 1992). Slow inactivation differs from fast inactivation in that it is relatively insensitive to cytoplasmic manipulations that have profound effects on fast inactivation. For example, fast inactivation is readily abolished by intracellular enzymatic treatment (Rojas and Armstrong, 1971; Quandt, 1987) and by mutations in the cytoplasmic loop linking the third and fourth domains of the Na 1 channel protein (Stühmer et al., 1989; Patton et al., 1992; West et al., 1992). These treatments do not disrupt slow inactivation (Rudy, 1978; Quandt, 1987; Valenzuela and Bennett, 1994; Cummins and Sigworth, 1996 b ). The molecular nature of slow inactivation and the location of its gate are unknown. We show here that both permeant and impermeant alkali metal cations in the extracellular solution affect the kinetics and steady-state levels of slow inactivation when compared with four different organic cations. These effects of alkali metal cations can be explained by their influence on two rate constants, one for entry into and one for recovery from a slow-inactivated state.