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Figure 4 Steady-state inactivation of I_to and I_Ca and inactivation properties of I_to in myocytes from 9- to 12-month-old nontransgenic (Ntg) and transgenic (Tg) mice. Representative current tracings for voltage-dependence of steady-state inactivation of I_to are displayed from Ntg and Tg cardiac myocytes (A, right panel). Voltage dependence of I_to inactivation was assessed using a double-pulse-protocol (A, left panel). The data are normalized to the largest peak I_to and I_Ca currents measured using a prepulse of –100 mV versus –80 mV (I_to/I_max or I_Ca/I_max). The total peak outward current includes a steady-state component that may not represent I_to. Data were fit to Boltzmann equation as follows: I = I_max/1+exp (V_m-V_0.5)/k, where I is recorded current, I_max is maximum current, V_m is membrane potential, V_0.5 is potential where inactivation is 50%, and k is the slope factor. Mean voltage-dependent inactivation curve of I_Ca for Ntg and Tg myocytes, respectively (B). Inactivation decay courses of I_to recorded during 680 ms depolarizing voltage steps to test potential +40 mV. The time courses were determined in individual Ntg and Tg cells (9- to 12-month-old) using the double exponential fits (using the Chebyshev algorithm of CLAMPFIT) to the decay phases of the currents (C and D) by the equation I(t) = A_fast[exp(–t/_fast] + A_slow[exp(–t/_slow)] + A: A_fast and A_slow being the maximal amplitude; _fast and _slow being the time constants of the fast and slow components of inactivation, respectively. Time zero was set next to the peak of the outward current. C and D show the _fast and _slow components of I_to inactivation at different depolarization potentials in Ntg and Tg cardiac myocytes. Continued on next page. (E and F) Superimposed original current recordings showing time-course of recovery from inactivation of I_to in Ntg and Tg cells. Capacitive transients were partially removed for clarity. (G) Plots showing reactivation of I_to in Ntg and Tg myocytes. Both curves were well-fitted by double exponential function.