S4 Charges Move Close to Residues in the Pore Domain during Activation in a K Channel

Abstract

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 Å) to a position close (8 Å) 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.

Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

“…29,30 This approach has been used to measure the electrostatic potential inside ion channel pores and crevices. [23][24][25][26][27]29 The relative rates of modification of a given cysteine in a protein by positively and negatively charged reagents relative to the ratio of these rates for a free thiol in solution, e.g. β-mercaptoethanol (βME), is an exponential function of the electrostatic potential (Ψ).…”

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1

“…Inherent in the strong screening hypothesis is the assumption that Sr 2þ has less effect on the channel during the final step than during the early steps. It has been shown for Shaker channels that j out close to S4 changes by þ35 mV when the channel changes from a closed state with fully retracted gating charges to an open state with the gating charges exposed on the extracellular surface (54). Thus, j out as determined from Eq.…”

Voltage-Sensor Transitions of the Inward-Rectifying K+ Channel KAT1 Indicate a Latching Mechanism Biased by Hydration within the Voltage Sensor

“…The Kv1.2 and Shaker channels open with positive-going voltages that drive the VSD into the up state. This state exposes residues of the S4 a-helix otherwise buried within the membrane to modification by aqueous Cys-reactive, methanethiosulfonate reagents from the outside and hides others on the inside on the channel opening (Larsson et al, 1996), and it aligns key residues in the S4 a-helix in close proximity with amino acids near the outer surface of the S5 a-helix (Elinder et al, 2001). The same conformation in the KAT1 VSD has been associated with the closed channel (Latorre et al, 2003;Lai et al, 2005): for example, residues in the S4 a-helix move inward toward the cytosol (the down position), becoming inaccessible to the same membrane-impermeant reagents outside when the channels open at negative-going voltages (Latorre et al, 2003), and amino acids that pack against the S5 a-helix at negative voltages in the open conformation of KAT1 correspond with residues in the Shaker and Kv1.2 K + channels that are similarly positioned but in the closed channel conformation (Lai et al, 2005).…”