S4 Is the Primary Voltage Sensor

S4 Sequence and Charge Pairing

A high-resolution structure of the voltage-sensing domain of an ion channel has yet to be obtained; however, some things are already known about the structure of this domain. S1 to S6 form transmembrane helices [8,13,14,19,25,33]. The S4 helix is conserved across voltage-gated cation channels and contains a positively charged arginine (R) or lysine (K) at every third position (Fig. 4). Because S4 spans the membrane, some of these charges sense the electric field. The spacing of positive charges creates a left-handed positively charged spiral (resembling a barbershop pole) along the length of the S4 helix. Three conserved negative charges, two in S2 and one in S3 (Fig. 1A), interact with the charges in S4 [35,36], suggesting that three positive charges of S4 could reside in the gating canal at one time. Given the gradual pitch of the spiral, three consecutive positive charges would lie on the same face of S4.

S4 Positive Charges Account for the Gating Charge

Wild-type Shaker channels move ~ 12 to 13 charges per channel (~ 3 charges per subunit) during activation [28]. If S4 is the voltage sensor, then a mutation that neutralizes a positive

Electrical Voltage Ion Channels

Figure 2 Electrical properties of simulated ion channels. (A) Plot of the movement of the gating charge (Q) and the probability of the channel opening (Po) at various voltages. Because all voltage sensors must be activated before a channel opens, Q precedes Po in voltage dependence. (Modeled as in Ledwell and Aldrich [18].) (B) Gating current and ionic current elicited by depolarization. Movement of the voltage sensor generates several nanoamperes of gating current, opens the pore gates, and allows several microamperes of ionic current to flow through the pore.

Figure 2 Electrical properties of simulated ion channels. (A) Plot of the movement of the gating charge (Q) and the probability of the channel opening (Po) at various voltages. Because all voltage sensors must be activated before a channel opens, Q precedes Po in voltage dependence. (Modeled as in Ledwell and Aldrich [18].) (B) Gating current and ionic current elicited by depolarization. Movement of the voltage sensor generates several nanoamperes of gating current, opens the pore gates, and allows several microamperes of ionic current to flow through the pore.

charge on S4 should decrease the total gating charge. The maximum reduction expected is 1 charge per subunit for a position that completely traverses the electric field. Of the seven positive charges in the Shaker S4, single neutralizations of R1, R2, R3, or R4 each decrease the total charge by ~ 1 to 1.7 charges per subunit, whereas neutralization of K7 has no effect [1,32]. This indicates that only the outer S4 charges move through the gating canal. In addition, neutralization of the deep negative charge in S2 also decreases the gating charge by ~1.5 charges per subunit, suggesting that it may move across the electric field in a direction opposite to that of S4 [32]. The fact that some neutralizations decrease the charge by >1 indicates that a neutralization may affect the remaining charges, possibly by changing the pointing angle of their side chains and/or the shape of the electric field in the gating canal.

Residues in S4 Move Fully Across the Membrane

A direct measure of transmembrane motion in S4 has been obtained in Na+ and K+ channels by probing the accessibility of engineered single cysteines to internal and external thiol-specific reagents [3,16,37,38,39,40,43]. In the Shaker K+ channel, the charged and uncharged positions from R1 to R3 are inaccessible to the external solution at negative voltage (resting state) but are accessible to the external solution at positive voltage (activated state). R3 and deeper sites are accessible to the internal solution at negative voltage but inaccessible at positive voltage. The change in accessibility can be accounted for by a rigid body motion of S4 across the membrane (Fig. 4). The motion takes place in multiple steps with at least one intermediate position [3,4]. The model of transmembrane S4 motion is supported by the finding that histidines substituted at positions R2, R3, or R4 can transport protons across the membrane at voltages that allow the voltage sensor to shuttle between resting and activated states [34].

Taking into account all of the accessibility results leads to the following conclusions:

1. S4 moves outward with depolarization in the correct direction to carry the gating charge.

Figure 3 Three ways to move charge through the membrane. (A) Two charged membrane-spanning protein faces slide past each other to move one positive charge across the membrane. (B) A positive membrane-spanning segment slides through adjacent protein and interacts with negative counter charges. Water-filled vestibules formed by the protein shorten the distance required to move one positive charge across the membrane. (C) Movement of K+ ions through the short KcsA selectivity filter.

Figure 3 Three ways to move charge through the membrane. (A) Two charged membrane-spanning protein faces slide past each other to move one positive charge across the membrane. (B) A positive membrane-spanning segment slides through adjacent protein and interacts with negative counter charges. Water-filled vestibules formed by the protein shorten the distance required to move one positive charge across the membrane. (C) Movement of K+ ions through the short KcsA selectivity filter.

Protein Counter

Figure 4 Helical screw motion of S4 activation. (A) Depiction of S4 (the screw) moving through a short gating canal surrounded on either end by two water-filled crevices. Positive charges on S4 interact with negative counter-charges in the adjacent protein (the bolt). The water crevices hydrate S4 charges that do not interact with counter-charges. Depolarization rotates S4 along the stripe of charged positions and in the process moves S4 outwards along its helical axis. (B) Helical net of S4 that illustrates changes in solvent exposure and movement of charge across the gating canal. Positions R1, R2, R3, and R4 move some distance across the gating canal when S4 changes conformation from the resting to activated state.

Figure 4 Helical screw motion of S4 activation. (A) Depiction of S4 (the screw) moving through a short gating canal surrounded on either end by two water-filled crevices. Positive charges on S4 interact with negative counter-charges in the adjacent protein (the bolt). The water crevices hydrate S4 charges that do not interact with counter-charges. Depolarization rotates S4 along the stripe of charged positions and in the process moves S4 outwards along its helical axis. (B) Helical net of S4 that illustrates changes in solvent exposure and movement of charge across the gating canal. Positions R1, R2, R3, and R4 move some distance across the gating canal when S4 changes conformation from the resting to activated state.

2. S4 carries the equivalent of 3 charges per subunit across the membrane (R1 to R4 carrying 0.5, 1, 1, and 0.5 charges, respectively), accounting for the total gating charge in wildtype channels.

3. Only a short length of S4 (10 residues, ~ 13.5 Â) lies in the gating canal at any one time, which means that the canal is considerably shorter than the 35 Â thick core of the membrane and that the electric field is focused on S4.

S4 Moves at the Right Time To Generate the Gating Current

The contribution of S4 charges to the gating charge and the measure of transmembrane S4 motion argue that S4 is the primary voltage sensor. To prove this, it is necessary to show that S4 motion occurs during gating charge movement. The kinetics of protein motion in a channel can be measured optically using voltage-clamp fluorometry (VCF). Fluorophores sensitive to their local environment report local structural rearrangements with a change in fluorescence intensity [24]. The fluorescence of a probe attached at or near S4 changes brightness at voltages where channels do not open but where gating charge moves. The fluorescence and gating charge correlate both kinetically and in steady-state voltage dependence (Fig. 5) [6,9,23,24].

S4 moves in the right direction by a sufficient amount and with the correct kinetics for it to generate the gating charge. But how does it move? Fluorescence resonance energy transfer (FRET) between donor-acceptor fluorescent probes attached to S4s of different subunits provides a clue. An examination of the pattern of voltage-driven distance changes between S4s suggests that S4 twists in an 180° rotation [7,10]. Thus, the motion of S4 appears to involve both outward and rotary components.

Figure 5 Optical measurements demonstrate that S4 movement generates the gating current. Gating current (Ig), gating charge (Q), and fluorescence (F) were measured simultaneously from a single oocyte expressing nonconducting Shaker channels labeled at either position 350 or 359 with rhodamine maleimide (TMRM). The probe shows voltage-dependent changes in fluorescence intensity for which the kinetics correlate with gating charge movement. The fit to Q is overlaid over the fluorescence trace. For reference, position 359 is located 3 residues before R1 (see Fig. 4).

Figure 5 Optical measurements demonstrate that S4 movement generates the gating current. Gating current (Ig), gating charge (Q), and fluorescence (F) were measured simultaneously from a single oocyte expressing nonconducting Shaker channels labeled at either position 350 or 359 with rhodamine maleimide (TMRM). The probe shows voltage-dependent changes in fluorescence intensity for which the kinetics correlate with gating charge movement. The fit to Q is overlaid over the fluorescence trace. For reference, position 359 is located 3 residues before R1 (see Fig. 4).

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