|
Figure 1. Activation and Deactivation Characteristics.A) Main Panel: Comparison of normalized (peak values at +50 mV) mean peak transient I–V relationships for V287R (n = 8) and WT Kv4.3, from [16]. HP = −100 mV, currents elicited by one second depolarizing pulses, and mean peak transient current defined as peak current minus residual current at the end of the depolarizing pulse. V287R mean peak current amplitude at +50 mV = 903±141 nA. Inset: Representative current waveforms for V287R elicited in response to depolarizing voltage clamp pulses applied from −10 mV to +50 mV, 10 mV increments. Calibration bar: 200 nA, 50 ms. B) Mean steady-state activation curve “a4” fit as a fourth order Boltzmann relationship (V1/2 = 1.40 mV, k = 11.62 mV, n = 11). Mean WT “a4” relationship: V1/2 = −36 mV, k = 14.50 mV, from [9]. C) Main Panel: V287R activation kinetics (n = 11). Curve fits (single exponential functions) to mean data points: τact = 4.84e10−mV/30.9+0.20 ms. Inset: Representative fits of V287R “a4” activation kinetics elicited during depolarizing pulses from −10 to +50 mV, 10 mV increments, HP = −100 mV. τact = 1.1, 1.4, 1.9, 2.5, 4.2, 5.2, 7.7 ms, −10 to +50 mV, respectively. Calibration bar: 200 nA, 1.0 ms. D) Main Panel: V287R deactivation kinetics (n = 12). Curve fits (single exponential functions) to mean data points: τdeact = 0.18emV/32.7+1.40 ms. Inset: Representative single exponential fits of V287R deactivation kinetics from −40 to −100 mV, 10 mV increments, HP = −100 mV. τdeact = 4.9, 3.9, 3.6, 2.7, 2.8, 2.2 ms, −40 to −100 mV, respectively, −70 mV not fit. Calibration bar: 50 nA, 3.0 ms. WT data in panels C) and D) from [9].
|
|
Figure 2. Inactivation Characteristics.A) Isochronal one second inactivation relationship “i”. Mean data points (n = 13) fit to a single Boltzmann relationship (V1/2 = −51.50 mV, k = 7.26 mV). WT data (V1/2 = −60.10 mV, k = 6.20 mV) from [9]. B) Comparative overlays of “a4” and “i” relationships for WT and V287R channels. Both expression conditions displayed significant closed-state inactivation, with V287R stabilizing non-inactivated closed states. The mutant channel also produced a greater depolarizing shift in “a4” than in “i”. C) V287R mean τinact−Vm curves (n = 12). Curve fits: τfast = 86.20e10−mV/12.23+23.50 ms; τslow = 99.70e10−mV/33.97+201.90 ms. WT data from [10]. D) Main Panel: V287R kinetics of closed-state inactivation. A fixed one second pulse to +50 mV (P2) was preceded by a pulse of progressively increasing duration (P1) at each of the potentials indicated. The decline of P2 current as a function of P1 duration was used to determine τcsi. V287R curve fit: τcsi = 725.68e−60+mV/10.23+91.50 ms. WT data from [25]. Inset: Development of CSI at −40 mV for V287R, τcsi = 202 ms. Calibration Bar: 100 nA, 150 ms.
|
|
Figure 3. Recovery Characteristics.A) WT and V287R macroscopic recovery kinetics (HP = −100 mV) developed during a one second pulse to +50 mV. Mean mutant data points (n = 7) fit as a single exponential function with τrec = 30.10 ms. WT τrec = 206 ms, data from [9]. B) Main Panel: Comparison of the voltage dependence of mean τrec values of V287R and WT, from [9], [10], [16], [25]. At all HPs, macroscopic recovery kinetics were significantly faster for V287R than for WT. Curve fits: WT: τrec = 23.89emV/11.14+142.10 ms, V287R: τrec = 9.42emV/21.77+13.95 ms. Inset: Representative macroscopic recovery waveforms for V287R (P2 currents at +50 mV). Peak data points fit with a single exponential relationship with time constant = 32 ms. Calibration bar: 200 nA, 20 ms. C) Representative recordings of recovery (HP = −85 mV) from closed-state inactivation for WT (upper panel, fit as an “a2” formulation) and V287R (lower panel, exponential fit). Calibration bars: WT: 1 µA, 50 ms, V287R: 500 nA, 50 ms. D) Voltage dependence of mean τrec,csi values for WT (hollow squares, n = 5) and V287R (solid squares, n = 7). Curve fits: WT: τrec,csi = 248.50emV/93.1−582.10 ms, V287R: τrec,csi = 5.80emV/19.77+34.10 ms.
|
|
Figure 4. Summary of all Kv4.3 S4 mutant data collected to date: “i” V1/2 values as a function of “a” V1/2 values.The solid black line (centered on WT) is a linear relationship with slope = 1.0, as predicted from previous work on Shaker channels [26]. The solid gray line is a best fit to all mutant data points (WT excluded, slope = 0.65, mean R→A/Q data points from [9], [10]). All ΔV1/2 shifts were less than those predicted from Shaker.
|