J Gen Physiol
July 1, 2001;
S4 charges move close to residues in the pore domain during activation in a K channel.
Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.
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Figure 1. Transmembrane topology of the Shaker K channel. In the present study, we investigate the electrostatic interactions between residue 362 and residues 418 and 419.
Figure 2. Effect on the local concentration of charged MTS reagents and protonation state of a cysteine due to a change in the local electrostatic potential around the cysteine. (A) The movement of the positive charges of S4 toward the cysteine will increase the local concentration of MTSES− around the cysteine in the open state. The probability of finding the cysteine in the unprotonated reactive state will also increase in the open state. (B) The activation movement of S4 decreases the local concentration of MTSET+ around the cysteine in the open state. But, the probability of finding the cysteine in the unprotonated reactive state will increase in the open state.
Figure 3. Time course of reaction between MTSES− (A and C) or MTSET+ (B and D) and 418C channels (A and B) or 362Q/418C channels (C and D). Application of MTS reagent in the resting state (closed squares, −80 mV) or the activated state (open circles, 0 mV for 418C, and +40 mV for 362Q/418C). The fitted curves are single exponential curves with time constants in the resting and activated states: (A) 52.3 and 2.8 mMs, (B) 3.0 and 1.5 mMs, (C) 40.4 and 13.2 mMs, and (D) 1.5 and 0.9 mMs.
Figure 4. The mutation R362Q does not alter the inactivation kinetics. The voltage is +40 mV during the first 20 s. Then, the membrane voltage is reset to −80 mV for variable times followed by a short voltage pulse to +40 mV. The current scale (ordinate) has been normalized to give equal magnitudes for the initial current peaks for both WT and R362Q.
Figure 5. Model of conformational changes during activation and slow inactivation. The structure of the KcsA channel has been used as a template when constructing this cartoon, keeping the distances between residues as in the KcsA channel (Doyle et al. 1998). S4 is shown with solid lines in the activated state and dashed lines in the resting state. (A) Side view (see arrows in B) of the channel (∼20° above the plane of the lipid bilayer). Only two opposite subunits are shown. The K ions in the selectivity filter are the closed circles and the water molecule is open. (B) Extracellular view of the channel. Residues in one subunit are labeled. For simplicity, only S4 in this subunit is shown in its tilted position. Note that the loop from S5 to the center of the channel is only schematically drawn. (1) Upon a positive change in membrane potential S4 moves outward and rotates 180° (Keynes and Elinder 1999). (2) S6 rotates 15° and opens the activation gate (Perozo et al. 1999). (3) Residue 451 rotates toward 418, and 450 is removed from its position in the aromatic cuff (Larsson and Elinder 2000). (4) The aromatic cuff (W434, W435, and Y445) can constrict and the pore is closed at the selectivity filter (backbone of Y445).
Contribution of the S4 segment to gating charge in the Shaker K+ channel.