Bao H et al. (1999), Voltage-insensitive gating after charge-neutral...

XB-ART-13739

J Gen Physiol 1999 Jan 01;1131:139-51.

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Voltage-insensitive gating after charge-neutralizing mutations in the S4 segment of Shaker channels.

Bao H , Hakeem A , Henteleff M , Starkus JG , Rayner MD .

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Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from -160 to +80 mV with a constant opening probability (Po). Although Po is low ( approximately 0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca2+ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb+ is substituted for K+ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive Po seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.

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	Figure 1. Voltage sensitivity of gating in mutants 12Q, 127Q, 24Q, and 147Q, compared with ShΔ. (A) Macroscopic currents from inside-out patches in steps from −80-mV holding potential to −30 and +80 mV, respectively, returning to −100 mV. Leak holding potential was −120 mV. Note, similar kinetics of activation and deactivation for 12Q, 127Q, 147Q, and ShΔ. (B) Macroscopic currents for the 24Q mutant. Due to the marked left shift in Po(V) curve for this mutant (C), holding potential was −160 mV for this mutant and currents at four test potentials are shown here (see pulse protocol insert). At −140 mV, no significant current is seen. Inward currents occur at −100 and −20 mV, with outward current at +60 mV. Leak holding potential was −160 mV. (C) Normalized voltage dependence of channel open probabilities for 12Q, 127Q, 24Q, and 147Q, as well as for control ShΔ.
	Figure 3. Typical single channel current traces for 124Q (left) and 1247Q (right) in symmetric 115 mM K+ solutions at various test potentials. Both mutant channels open across the voltage range from −160 to +80 mV. Holding potential was 0 mV for both mutants.
	Figure 5. Analysis of single channel currents from the 1247Q mutant at −40-mV test potential in symmetric 115-mM K+ solutions (A), and in external 115-mM Rb+ solution (B). For each solution, single channel amplitude histograms are shown (top) together with logarithmic open time (bottom) and closed time distributions (center). Lines of fit were obtained using a maximum likelihood method. Dashed lines indicate individual exponential components. (C) Open time constants and the four closed time constants are plotted as functions of test potential, data from 124Q (open symbols), and from 1247Q (solid symbols). The mean open time for wild-type ShΔ is indicated as open square at the voltage of 20 mV. The three closed time constants for wild-type ShΔ are indicated as the dashed line. Data in A and B were from two representative patches. Data in C are means.
	Figure 4. Po(V) relationships for the 124Q and 1247Q mutants, compared with wild-type ShΔ channels. The wild-type ShΔ data were derived from Fig. 1 A control ShΔ data, scaled to the observed Po,max at +80 mV for ShΔ channels. Single channel data were recorded in inside-out configuration and symmetric (115 mM) K+ solutions or Rb+ (115 mM)//K+ (115 mM) solutions as indicated. All data points are means ± SD, n = 3–6 patches.
	Figure 6. Pore properties of voltage-insensitive 124Q channels. (A) Unidirectional single channel currents measured at +40 mV (Tris Ringer//“X+” EGTA, shaded histograms) and at −40 mV (“X+” Ringer//Tris EGTA, open histograms). The histograms show the following permeability sequence: K+ > Rb+ > NH4+ > Cs+ and Na+. (B) 124Q channels are sensitive to block by 4-AP. I–V data points from one representative oocyte using two-electrode voltage clamp. Macroscopic current seen in 115-mM external K+ solution (▴) appears effectively blocked after addition of 10 mM 4-AP to the bath solution (○).
	Figure 7. The 3+2′ model redrawn from Schoppa and Sigworth (1998c). Estimated reaction valences from their model fits to wild-type ShΔ channel data are shown above each voltage-sensitive reaction step. Shaded areas are presumed to be dysfunctional in voltage-insensitive 124Q and 1247Q mutant channels.

References [+] :

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