In summary the data support the following conclusions: 1. Phe233 forms the extracellular lid on an occluded binding site in voltage sensors into which S4 positive charges bind. 2. Mutating Phe to Trp alters the relative strength of interaction between Arg or Lys and this site. 3. Lys appears to interact more favorably compared to Arg in the presence of Trp: at position 5 Lys stabilizes the open voltage sensor conformation, at position 1 Lys stabilizes the closed voltage sensor conformation. 4. Given that Lys at position 1 and at position 5 appears to interact with the site in the closed and open conformations, respectively, we propose that the occluded site serves as a catalytic center to lower the energy associated with the transfer of each of the voltage sensor charges as they cross the membrane. 5. Arg at position 5 isolates the last 25% of gating charge as a separate component in the Q–V plot, which occurs concomitantly with pore opening. The association of this last component of gating charge with pore opening suggests that binding of the charged residue at position 5 into the occluded site is required for pore opening.
A simple state model of voltage sensor transitions is shown (). Each of the five states, connected by four transitions, corresponds to conformations of a single voltage sensor associated with the positive charged residue at positions 1 through 5 inside the occluded site. Thus, state 1 represents the fully closed voltage sensor in which the positive charge of the position 1 amino acid binds to the occluded site, and state 5 represents the open voltage sensor in which the positive charge of the position 5 amino acid binds to the occluded site. The data do not uniquely define each transition in this model so the following simplifying assumptions are made. A single value for the forward rate constants and a single value for the backward rate constants are used except for the rate constants describing the fourth transition connecting states 4 and 5. Voltage-dependence is distributed over the four transitions equally, with 25% of the total gating charge in each. It is further assumed that the open pore corresponds to the condition in which all four voltage sensors are open (i.e. all four have reached state 5.) and that four voltage sensors undergo independent conformational changes.
For illustrative purposes the graphs below the state model depict chemical free energy as a function of voltage sensor reaction coordinate in a channel in which Phe has been mutated to Trp (). The presence of Lys in the occluded site is represented as a deeper energy well relative to Arg. One physical interpretation is that Lys binds more tightly to the occluded site. Another interpretation is that the energy barriers and wells are higher for Arg relative to Lys. Equations for the scheme in (Supplementary Information
) generate the main features observed in the electrophysiology data (). Notably, by changing only the well depth of state 5 by approximately 4.6 RT relative to all other barriers and wells, the qualitative features of both the gating current time course and the Q–V plots of R1K5(W) and R1R5(W) are recapitulated (). In particular, relative stabilization of state 5 with Lys in the presence of Trp [R1K5(W)] prolongs the gating current associated with channel closing and gives it the correct time-dependent shape (), whereas destabilization of state 5 with Arg in the presence of Trp [R1R5(W)] speeds the gating current and separates the Q-V plot into two components ().
The model captures an important feature of gating, most explicitly displayed in channels with Arg at position 5 [R1R5(W) and K1R5(W)]: multiple transitions occur within each voltage sensor (connecting states 1 through 4 in the model) prior to a final transition (state 4 to 5), which is closely associated with pore opening (, and ). Thus, according to the model, the closed channel is actually associated with a distribution of conformations (states 1 through 4 within each voltage sensor), which is a function of the degree to which the membrane is hyperpolarized: the more negative the voltage, the closer the distribution comes to state 1. The open channel on the other hand is associated with a specific conformation (state 5) that must be achieved by all four voltage sensors. In order to approximate the behavior of the electrophysiology data we found it necessary to use unique forward and backward rate constants for the fourth (final) voltage sensor transition (Supplementary Information
). We speculate that this final transition is very different because it is associated with opening the pore.
In the context of the crystal structure the model can explain a discrepancy between the total gating charge per channel (approximately 14 elementary charges) and the sum of reduced charges when each of the five positions is mutated to a neutral amino acid one at a time (approximately 18 elementary charges) (6
). Due to the presence of two carboxylate ligands, the occluded binding site probably requires a positive charge in it at all times – except during brief transitions between states driven by voltage. Therefore, mutations that remove a positive charge from a particular position should reduce measured gating charge for two reasons: because the number of charges transported by the voltage sensor is reduced, and because the distribution of states before and/or after the voltage step is altered (i.e. the starting and/or ending positions of all the charges is altered). The two reasons combined can account for the apparent discrepancy between total gating charge and the sum of reduced gating charge by mutation.
We do not know the physical basis of Lys versus Arg discrimination by the occluded site in the Phe to Trp mutant. The case in which Lys binds more favorably to the occluded site is indistinguishable from that in which Arg binds less favorably because the Boltzmann distribution of states depends only on the relative energy differences among the available states. We favor the idea that Trp causes Arg to bind in the site with lower affinity, and also perhaps raises the barrier for an Arg to enter the site because Trp is larger than Phe and would be expected to constrict the site, destabilizing the larger Arg guanidinium group relative to the smaller Lys amino group. A correlation between the size of substituted groups on the Phe side chain (Br > CN > ME > H) and the value of Vm is consistent with this idea (fig. S1
The data imply a specific distance over which S4 charged amino acids move across the membrane with gating. In the crystal structures of the open conformation Lys at position 5 is located in the occluded binding site ( and ) (11
). The electrophysiological data suggest that Lys at position 1 binds in the occluded site in the fully closed (strongly hyperpolarized) conformation (). The α-carbon distance between position 1 and 5 is 21 Å along the S4 helix and 18 Å perpendicular to the membrane (fig. S4
). This distance falls within the range 15 Å to 20 Å inferred independently by biotin-avidin accessibility studies on a prokaryotic Kv channel (18
This study identifies an occluded site, conserved in voltage sensors, that catalyzes the transfer of positive charges across the membrane field in the process of voltage sensing. By manipulating the structure of the site we have altered its selectivity between Arg and Lys. This selectivity is used to stabilize the voltage sensor in specific conformations and dissect the relationship between the voltage sensor and pore conformational changes.