Mathur R et al. (1997), Role of the S3-S4 linker in Shaker potassium ch...

XB-ART-16976

J Gen Physiol 1997 Feb 01;1092:191-9. doi: 10.1085/jgp.109.2.191.

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Role of the S3-S4 linker in Shaker potassium channel activation.

Mathur R , Zheng J , Yan Y , Sigworth FJ .

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Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge change, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three-to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels.

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Species referenced: Xenopus laevis
Genes referenced: kcnc2 tbx2 xa-1

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	Figure 7. Activation and deactivation in chimeric channels. (A) Time courses of activation during a âˆ’20-mV depolarization for ShÎ”, ShÎ”/Shal, ShÎ”/Shaw, and ShÎ”/Shab currents, and for the cysteine insertion mutant ShÎ”/Cys. Currents in the mutant constructs were scaled by factors of 0.9, 5.0, 1.0, and 2.17, respectively, to match the ShÎ” currents. (B) Deactivation time courses at âˆ’60 mV after a prepulse to +40 mV. Currents from ShÎ”/Shal, ShÎ”/Shaw, and ShÎ”/Shab were scaled by factors of 1.5, 0.78, 1.45, and 1.25, respectively. (C) Voltage dependence of the predominant time constant of deactivation, Ï„d1, as a function of test potential for ShÎ” and the chimeric channels. Plotted are the geometric meansâ€‚Â± SE (n = 6).
	Figure 2. Mutations of the Shaker S3-S4 region. The wild-type Shaker sequence (top) is compared with the sequences of mutations studied here. Mutations included the deletion and charge-reversal of acidic residues at the NH2-terminal end, deletions at the COOH-terminal end, and substitutions of each of the three proline residues with an alanine. In the insertion mutations (ShÎ/Cys, ShÎ/FLAG, ShÎ/HA, ShÎ/Xa) the underlined sequences were inserted following proline 344, as indicated by the upward arrows. In the final three mutations, the entire S3-S4 linker was replaced with shorter segments from Shab11, Shaw2, or Shal2, respectively (Butler et al, 1990). Dashes indicate amino acid identity to the Shaker sequence and blank spaces indicate gaps.
	Figure 3. Whole-oocyte voltage-clamp protocols and currents from oocytes expressing the âwild-typeâ ShÎ and the ShÎ/Shaw chimeric channels. (A) Activation protocol. Depolarizations of 100-ms duration are given from a holding potential of â80 mV. (B) Deactivation protocol. After a 20-ms prepulse to +40 mV, tail currents are measured during 100-ms test pulses at potentials between â100 and +40 mV. Potentials are varied in steps of 10 mV, and pulses are applied at 5-s intervals.
	Figure 4. Voltage dependence of activation and time constants. Normalized conductance-voltage (G-V) curves, and voltage dependence of activation time constant are shown for ShÎ” and various mutants: (A) deletions at the NH2-terminal end of the linker; (B) charge reversals at the NH2-terminal end; (C) deletions at the COOH-terminal end. G-V curves (top) are shown for individual oocytes, chosen to have Va and ka values near the medians of 4â€“10 recordings. The conductance values were computed from the average current during the second half of 100-ms depolarizations to the given voltages and are shown fitted to the fourth power of a Boltzmann function. Time constant Ï„a1 of the predominant component of the activation time course (bottom) is plotted as the geometric mean Â± SE for n = 5â€“7 determinations as a function of depolarizing voltage. Lines connect the values for ShÎ” (filled circles).
	Figure 5. Comparison of the time course of activation of ShÎ and the ÎA and ÎAMS mutants. Depolarizations to â20 mV were applied from a holding potential of â80 mV; the entire time courses are shown in the inset. The ÎA and ÎAMS current recordings were scaled by factors of 1.57 and 2.54 to match the steady-state current in the ShÎ recording.
	Figure 6. Voltage dependence of activation and time constants of mutants. (A) Proline substitutions; (B) insertions; (C) linker sequence substitutions. G-V curves (top) and time constants (bottom) are plotted as in Fig. 4. Error bars represent standard errors with nâ= 5â7.
	Figure 8. Determination of apparent gating valence zapp. (A) Conductance-voltage curve. Mean current from ShÎ channels during the second half of 400-ms depolarizations from â80 mV were converted to conductance and plotted as open circles as a function of voltage; conductances obtained from shorter (100 ms), larger depolarizations are plotted as filled circles. (B) Values of zapp, computed as the logarithmic derivative of G with respect to voltage according to Eq. 2 are plotted as a function of G as in Zagotta et al. (1994). Because of growing errors at lower conductances, we took the estimate of zapp to be the value at 1% of the maximal conductance, as indicated by the arrow.

References [+] :

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