Broomand A et al. (2003), Molecular movement of the voltage sensor in a K...

XB-ART-4435

J Gen Physiol 2003 Dec 01;1226:741-8. doi: 10.1085/jgp.200308927.

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Molecular movement of the voltage sensor in a K channel.

Broomand A , Männikkö R , Larsson HP , Elinder F .

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The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 A) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 A) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.

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Species referenced: Xenopus laevis
Genes referenced: tbx2

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	Figure 1. . Formation of disulphide bonds between residues in S4 and residues in the pore domain. (A) Sequences of the proposed voltage sensor paddle (Jiang et al., 2003a). α-Helical parts are underlined. S3b and S4 are packed to each other as suggested by Jiang et al. (2003a); that is, COOH terminus of S3b is to the right while COOH terminus of S4 is to the left. 22 residues of the Shaker S3-S4 linker are omitted. Positive charges in red and negative in blue. The net charge of the paddles: +3 (KvAP), +1 (Shaker), and 0 (RnorvEAG). (B) Molecular structure of S4 of KvAP (Jiang et al., 2003a) and the pore domain (S5-S6; side view to the left and top view to the right) of KcsA (Doyle et al., 1998). The residues used in the present investigation are labeled (Shaker numbering). The α-helical parts of S4, S5, and S6 in one subunit are space filled. The structure between 353 and 362 is not known for the Shaker K channel and is only drawn as a random coil. (C) Voltage-clamp data for six double-cysteine mutations. Current in response to a voltage step to 0 mV in control solution (1K), after incubation in 0.5 mM DTT (continuous line), and in control solution (1K), after >1 min exposure to Cu/phenanthroline (dotted line). Holding voltage was −80 mV. (Bottom) DTT (20 mM) reversed the Cu/phenanthroline effect, showing that Cu/phenanthroline induced disulfide bonds (shown for 359C/416C channels).
	Figure 2. . Voltage and time dependence of disulphide bond formation. (A) State-dependent formation of disulphide bonds between 362C and 416C for three consecutive voltage steps to 0 mV. Voltage protocol is shown in the top panel. Cu/phenanthroline was added for 20 s, as indicated. Applied at −100 mV, Cu/phenanthroline did not affect the current; applied at 0 mV, Cu/phenanthroline clearly reduced the current. (B and C) Steady-state current at the end of a 70-ms pulse to 0 mV versus accumulated exposure to 2/100 μM Cu/phenanthroline. The voltage was stepped to −80 mV for 20 s, then to either −80 mV or to 0 mV for 30 s, followed by a voltage step to −80 mV for 10 s. For each episode, Cu/phenanthroline was applied for a brief period (usually 5 s) during the 30-s step either to −80 mV or to 0 mV. The data points at time < 0 were obtained in the absence of Cu/phenanthroline. n = 3–7 for all data points. Eq. 1 was fitted to the data points, with w = 1 for the continuous curves and w = 4 for the dashed curve. (B) Disulfide bond formation in 362C/416C at −80 mV (open circles) and 0 mV (closed circles). Spontaneous current reduction (time < 0): τ(−80) = 104 min and τ(0) = 33 min (w = 1). Cu/phenanthroline-induced current reduction (time > 0): τ(−80) = 27 s and τ(0) = 10 s (w = 1). τ(−80) = 14 s for w = 4. (C) 353C/416C. Spontaneous current reduction (time < 0): τ(−80) = 18 min and τ(0) = 24 min (w = 1). Cu/phenanthroline-induced current reduction: τ(−80) = 18 s and τ(0) = 15 s (w = 1).
	Figure 3. . Disulphide bond formation between two subunits. Equal amounts of 359C and 416C mutations were coinjected in the oocytes. Cu/phenantroline was applied for 60 s at 0 mV. Current in response to a voltage step to 0 mV before Cu/phenanthroline application (continuous line) and after a 60-s application of Cu/phenanthroline at 0 mV (dotted line). Holding voltage was −80 mV.
	Figure 4. . Model for S4 location in the Shaker K channel (stereoview). To construct a structural model of the Shaker K channel, we used the KcsA structure (Doyle et al., 1998) for the pore and added the isolated S1-S4 structure of KvAP (Jiang et al., 2003a). The top of S4 is in close proximity to the top of S5 in this model. The four subunits of KcsA are colored red or gray. Only one of the four voltage-sensing S1-S4 domain is shown: S1 (blue), S2 (green), S3 (light blue), and S4 (dark red).
	Figure 5. . Model for S4 movement in the Shaker K channel based on X-ray crystallographic structures of related channels and data from the Shaker K channel. To construct structural models of the different channel states, we used the KcsA structure (Doyle et al., 1998) for the closed configurations of the pore and the MTHK structure (Jiang et al., 2002) for the open configuration. To these pore structures we added modified versions of the isolated S1-S4 structure (Jiang et al., 2003a). S4 in KvAP differs from most other voltage-gated K channels in having unequal spacing of the positive charges. We therefore realigned the Shaker and KvAP sequences to preserve the electrostatic interactions in the crystal structure. We assumed that the residues in the crystal structure of KvAP (133 and 136) interacting with negative charges in S2 (62 and 72) and S3 (93) are homologous to 371 and 374 in the open state of Shaker. Thus, to model S1-S4 in the open state, we assumed that 124–139 in KvAP corresponds to 362–377 in the Shaker channel. The residue corresponding to 362 in the Shaker channel is close to the residue corresponding to 416 in the neighboring subunit. To model the closed state, we moved S4 downward in a helical-screw fashion, so that 362, 365, and possibly 368 interact with the negative charges in S2 and S3.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase