Conti L et al. (2016), Reciprocal voltage sensor-to-pore coupling lead...

XB-ART-55128

Sci Rep 2016 Jun 09;6:27562. doi: 10.1038/srep27562.

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Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation.

Conti L , Renhorn J , Gabrielsson A , Turesson F , Liin SI , Lindahl E , Elinder F .

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Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd(2+) bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K(+) coordination, a hallmark for C-type inactivation. An engineered Cd(2+) bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

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	Figure 1. The pore domain/VSD interface is important for C-type inactivation.(a,b) Molecular structure of the Shaker Kv channel (top view in a, side view in b). Four identical voltage-sensor domains (VSDs) surround the pore domain. The voltage sensor S4 (positively charged residues in blue) is in an activated up-state. For clarity, the VSDs in the front and the back are removed (b). Labelled residues are discussed in this paper. (c–e) Effects of different mutations and pH on the inactivation time course and steady-state current. Test step voltage = +80 mV. Holding voltage = −80 mV. pH = 7.4 if not otherwise noted.
	Figure 2. Interactions between F416C of S5 and residues of S4 affect C-type inactivation.(a–e) 10 μM Cd2+ (red) affects the time course of slow inactivation in R362C/F416C (b) and R365C/F416C (c) but not in R359C/F416C (a) or R368C/F416C (d). Control in black. (e) 10 μM Cd2+ (red) slows down inactivation in R362C single mutant. (f) 10 μM Cd2+ (red) does not affect the inactivation time constant in E247Q/R362C. (g) 70 μM DHA (blue) accelerates inactivation in Shaker wt. (h) 70 μM DHA-me (blue) does not accelerate inactivation in Shaker wt. (i) 70 μM DHA (blue) only has a minor effect on inactivation in A359E/R362Q. For all panels, test step voltage = +80 mV, holding voltage = −80 mV, pH = 7.4.
	Figure 3. Interactions between S3 and S4 in the VSD affect C-type inactivation.(a) 10 μM Cd2+ (red) inactivates L327C/R368C to a lower steady-state current. Control in black. Test step voltage = +80 mV. Holding voltage = −80 mV. pH = 7.4. (b) Quotient of Cd2+ effects on the rate constants λ and κ calculated according to Scheme I (see Methods for details) for eight different channels. Error bars are calculated from the inverse of the data. n = 3–4. (b) Top view of model of the VSD and S5 of the Shaker channel based on a crystallographic structure. (d) Top view of the model based on our experimental data. Note how a Cd2+ bridge between L327C and R368C lifts R365 (cyan) above F416 (green) without moving more positive gating charges across the central hydrophobic barrier of the VSD.
	Figure 4. Molecular modelling of the mechanism for C-type inactivation.(a) Distances between the α carbons for six residues in the selectivity filter during a 300 ns long MD simulation when all four 416 residues are pulled away from the center of the channel at a rate of 0.001 nm/ns (see Methods for details). After 229 ns (vertical line) the distances are abruptly altered and the first K+ leaves the filter array. (b) A snapshot at the time of the filter collapse (229 ns) shows that the array of K+ in the selectivity filter retreat downwards. Water and Na+ interact with carbonyls (red sticks) in positions 445 and 446. Structure is aligned to initial frame of the simulation (grey sticks) with regard to N, CA, C, and O backbone atoms in position 441 to 446 of all four chains. (c–e) Filter distortion (top view). At positions 445 (c) and 444 (d) there is an increase in filter diameter (average of distances between carbonyls of opposing chains) compared to initial structure (grey sticks, dotted circle). At position 443 (e) there is a decrease in filter diameter. There is also side chain rotations of Y445 at the moment of filter collapse (c).
	Figure 5. Concerted action within a subunits during C-type inactivation.(a–h) Inactivation of eight different channels as denoted in the panels. Test step voltage = +80 mV; holding voltage = −80 mV; pH = 7.4. An inset is shown for T449A/K456M for a 20 ms long pulse (e). The pulses for T449V (g) and F416D/T449V (h) were 60 s long but for comparative reasons only 10 s are shown.
	Figure 6. F416C in the pore domain affects molecular motions within the VSD.(a) ON gating currents at 0 mV. Holding voltage VH = −80 mV. (b) Gating currents of F416C/W434F. VH = −80 mV. (c) Charge vs voltage of F416C/W434F calculated from ON gating currents. VH = 0 mV (black symbols), VH = −80 (white symbols). The continuous curves are best fits to Eq. 2 with a shared slope value. V½(−80 mV) = −61.0 mV, V½(0 mV) = −77.5 mV. The dashed curve is the best fit of a sum of two Boltzmanns’ curves (Eq. 2), where V½ for one component was fixed to −77.5 mV. V½ for the other component was determined to −58.3 mV. (d) OFF gating currents at −80 mV after 60 ms at 0 mV. (e) Gating currents of F416C/W434F. VH = 0 mV. (f) Charge vs voltage of W434F calculated from ON gating currents. VH = 0 mV (black symbols), VH = −80 mV (white symbols). The continuous curves are best fits to Eq. 2 with a shared slope value. V½(−80 mV) = −36.6 mV; V½(0 mV) = −64.9. All the recordings were done at pH 7.4.
	Figure 7. Mechanisms for C-type inactivation.(a) Molecular structure of one VSD (left) and the pore domain (right). Important residues explored in this study are space filled. Yellow double-directed arrow denotes approximate path for the molecular signal. (b) Postulated inactivation and Q(V)-shift pathways. Pore domain (blue), VSD (red), S4 (orange). Depolarization shifts the channel from a closed state to an open state. Prolonged depolarization either (1) rearranges the selectivity filter (inactivated state), and then alters the outer pore domain and S4 (inactivated* state), or (2) alters the outer pore domain and S4 (open* state), and then rearranges of the selectivity filter (inactivated* state).

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