Skerritt MR and Campbell DL (2008), Non-native R1 substitution in the s4 domain uni...

XB-ART-39095

PLoS One 2008 Jan 01;311:e3773. doi: 10.1371/journal.pone.0003773.

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Non-native R1 substitution in the s4 domain uniquely alters Kv4.3 channel gating.

Skerritt MR , Campbell DL .

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The S4 transmembrane domain in Shaker (Kv1) voltage-sensitive potassium channels has four basic residues (R1-R4) that are responsible for carrying the majority of gating charge. In Kv4 channels, however, R1 is replaced by a neutral valine at position 287. Among other differences, Kv4 channels display prominent closed state inactivation, a mechanism which is minimal in Shaker. To determine if the absence of R1 is responsible for important variation in gating characteristics between the two channel types, we introduced the V287R mutant into Kv4.3 and analyzed its effects on several voltage sensitive gating transitions. We found that the mutant increased the voltage sensitivity of steady-state activation and altered the kinetics of activation and deactivation processes. Although the kinetics of macroscopic inactivation were minimally affected, the characteristics of closed-state inactivation and recovery from open and closed inactivated states were significantly altered. The absence of R1 can only partially account for differences in the effective voltage sensitivity of gating between Shaker and Kv4.3. These results suggest that the S4 domain serves an important functional role in Kv4 channel activation and deactivation processes, and also those of closed-state inactivation and recovery.

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Species referenced: Xenopus laevis
Genes referenced: ap3b1 kcnd3 tff3.7

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	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.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase