Two atomic constraints unambiguously position the S4 segment relative to S1 and S2 segments in the closed state of Shaker K channel

Abstract

It is now well established that the voltage-sensing S4 segment in voltage-dependent ion channels undergoes a conformational change in response to varying membrane potential. However, the magnitude of the movement of S4 relative to the membrane and the rest of the protein remains controversial. Here, by using histidine scanning mutagenesis in the Shaker K channel, we identified mutants I241H (S1 segment) and I287H (S2 segment) that generate inward currents at hyperpolarized potentials, suggesting that these residues are part of a hydrophobic plug that separates the water-accessible crevices. Additional experiments with substituted cysteine residues showed that, at hyperpolarized potentials, both I241C and I287C can spontaneously form disulphide and metal bridges with R362C, the position of the first charge-carrying residue in S4. These results constrain unambiguously the closed-state positions of the S4 segment with respect to the S1 and S2 segments, which are known to undergo little or no movement during gating. To satisfy these constraints, the S4 segment must undergo an axial rotation of approximately 180 degrees and a transmembrane (vertical) movement of approximately 6.5 A at the level of R362 in going from the open to the closed state of the channel, moving the gating charge across a focused electric field.

Fig. 1.

The proton pore in the I287H mutant. (a) Current family recorded from an oocyte expressing the I287H mutant for a series of pulses from −160 to +60 mV in increments of 10 mV. The outside pH value was 5. (b) Average I–V relationship curve for the proton currents recorded from I287H mutants when the pH value was 5 (n = 5). (c) Same oocyte as in a but with outside pH value of 9. The currents in each potential were normalized to the maximal current. The holding potential was −90 mV and the test pulses (−160 to +60 mV) were preceded and followed by a −120-mV pulse. (d) Current family of another oocyte with an outside pH value of 5. (e) Same oocyte as in d after external addition of 5 mM Ni²⁺. Traces shown in d and e were subtracted by using P/4 protocol from a holding potential of +40 mV.

Fig. 2.

Effect of DTT and H₂O₂ on I287C-R362C mutant. (a) Currents recorded from an untreated oocyte. (b) Currents from the same oocyte after treatment with 10 mM DTT in the chamber. The holding potential was −90 mV and the 40-ms test pulse varied from −160 to 0 mV (P/−4 subtraction). (c) Average I–V curves measured at the end of 40-ms pulses from oocytes injected with I287C+R362C mutant before (open symbols) and after (filled symbols) treatment with 10 mM DTT (n = 3). The currents were normalized by the maximal current recorded after the treatment with DTT. The holding potential was −90mV, and the test pulses (−160 to +40 mV) were preceded and followed by a −120-mV pulse. (d) Gating and omega currents recorded from an oocyte expressing the I287C+R362C mutant and pretreated with DTT before mounting on the chamber. Dashed line indicates zero membrane current. (d and e) Currents recorded before (d) and after (e) external application of 10 mM H₂O₂. The records reflect currents elicited by pulses ranging from −160 to +40 mV with preceding and following pulses of −120 mV (off-line subtraction).

Fig. 3.

Effect of external Cd²⁺ on gating and omega currents of the I287C+R362C mutant. Pulses ranged from −160 to +40 mV in steps of 10 mV. (a) Control recording of an oocyte treated with DTT. (b) Recording after 1 μM Cd²⁺. (c) Recordings after 100 μM Cd²⁺. (d) Dose–response curve for the effect of Cd²⁺ on the omega current (filled circles) and the gating charge (open circles).

Fig. 4.

Recordings from the I241C+R362C mutant. (a) Currents recorded from an untreated oocyte. (b) Currents after treatment with 10 mM DTT in the chamber. (c) Currents from another oocyte that has been treated with 2 mM DTT. (d) Currents after external application of H₂O₂. (e) Currents from another oocyte that has been treated with DTT. (f) Currents after external application of 1 μM Cd²⁺. (g) Currents after external application of 100 μM Cd²⁺. Pulses ranged from −160 mV to +40 mV in a, b, and e–g. For traces shown in c and d, pulses from −150 to +40 mV were used. For traces shown in c–g, test pulses were not preceded or followed by a −120-mV pulse.

Fig. 5.

Minimal model of a Kv channel in its open and closed state. Only one voltage sensor and three subunits of the pore domain are shown for clarity. Segments S1 (white), S2 (yellow), and S4 (blue); the S4-S5 linker (slate blue); S6–S6 (green); and K⁺ ions (orange) are shown. The Cβ of the four gating charge of segment S4 (residues 362, 365, 368, and 371) are shown (magenta) together with the Cβ of I241 in segment S1 (white) and I287 in segment S2 (yellow). For the closed state, a Cd²⁺ (red) is placed where it would bind the cysteine residue of the V478C Shaker mutant. The dashed lines respectively indicate the center of the membrane and the membrane–solution interface at 0 Å and ±13 Å.