Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

Abstract

The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. One structural model predicts sequential formation of ion pairs between the S4 gating charges and negative charges in neighboring S2 and S3 transmembrane segments during gating. Here, we show that paired cysteine substitutions for the third gating charge (R3) in S4 and D60 in S2 of the bacterial sodium channel NaChBac form a disulfide bond during activation, thus ''locking'' the S4 segment and inducing slow inactivation of the channel. Disulfide locking closely followed the kinetics and voltage dependence of activation and was reversed by hyperpolarization. Activation of D60C:R3C channels is favored compared with single cysteine mutants, and mutant cycle analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation.voltage-sensing ͉ gating charge ͉ mutant cycle ͉ sliding helix V oltage-gated ion channels conduct electrical currents that are essential for a wide range of physiological processes including neuronal excitation, action potential conduction, synaptic neurotransmission, and muscle contraction. In prokaryotes, ion channels are necessary for pH homeostasis, chemotaxis, and motility. Voltage-gated ion channels respond to changes in membrane potential by opening and closing (''gating'') their ion-conducting pathway across the cell membrane. Gating is rapid, reversible, and steeply voltage-dependent, suggesting that charged gating particles associated with the channels move across the membrane in response to the electric field (1). Capacitative ''gating currents'' caused by these charge movements (2, 3) indicate that approximately 3-4 positive charges per voltage-sensing module move outward during gating of sodium or potassium channels (4-10). The primary structure of sodium channels (11) revealed four homologous domains containing six predicted alpha-helical segments (S1-S6) in each. Subsequently, the positively charged S4 segment was proposed to have a transmembrane position, serve as the voltage sensor that perceives changes in membrane potential, and move outward along a spiral path during channel activation to conduct the gating current and initiate a conformational change to open the pore (12, 13). A major thermodynamic obstacle to transmembrane movement of the S4 segment is stabilization of its positively charged amino acid residues in the membrane environment. The sliding helix model posits (12) that sequential formation of ion pairs with negatively charged amino acid residues in the S1, S2, and/or S3 segments serve to stabilize the S4 segments in the membrane and thereby catalyze their transmembrane movement. A detailed structural version of th...

Hydrophobic plug functions as a gate in voltage-gated proton channels

“…Therefore, we use G(V) curves to estimate the free-energy coupling between residues. Although the above approach assumes that the structure of the channel is not significantly changed by mutations and it is known that some mutations do interfere with the structure, several papers on double-mutant analysis involving charge neutralization in channels have proven very successful (18,25,26). The coupling energy between Arg258 and Glu201 is about 3.7 kcal/mol (Materials and Methods), whereas the interaction between Arg255 and Glu201 yields the slightly smaller value of 2.4 kcal/mol (Fig.…”

“…3B). To estimate the free-energy coupling between Arg255 or Arg258 and Glu201 or Asp222 quantitatively, we apply thermodynamic mutant cycle analysis (18,19). Recent theoretical analysis shows that the free-energy differences between the open and closed states is more accurately extracted from the gating charge versus voltage curves than from G(V) curves (24).…”

“…1B). For example, it has been shown that S4 residues in Kv and Nav channels are stabilized by forming ion pairs with acidic residues in the other TM domains of the channels and that these interactions change between closed and open states (17,18).…”

Tracking a complete voltage-sensor cycle with metal-ion bridges

“…1A) (2)(3)(4). The positive charges in S4 make salt bridges with negative countercharges on their move through the VSD (4)(5)(6)(7)(8). It has even been proposed that the VSD undergoes a conformational alteration following the opening, when the channel relaxes to an inactivated, that is closed, state (9).…”

“…1B, Upper) to alter the ion channel kinetics (7,8,22,23). Disulfide bonds, however, have the disadvantage that cysteines up to 15 Å apart can be caught in a bond (24).…”