Börjesson SI and Elinder F (2011), An electrostatic potassium channel opener targe...

XB-ART-44244

J Gen Physiol 2011 Jun 01;1376:563-77. doi: 10.1085/jgp.201110599.

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An electrostatic potassium channel opener targeting the final voltage sensor transition.

Börjesson SI , Elinder F .

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Free polyunsaturated fatty acids (PUFAs) modulate the voltage dependence of voltage-gated ion channels. As an important consequence thereof, PUFAs can suppress epileptic seizures and cardiac arrhythmia. However, molecular details for the interaction between PUFA and ion channels are not well understood. In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect. Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect. Because different voltage-gated K channels have different charge profiles, this implies channel-specific PUFA effects. The identified site and the pharmacological mechanism will potentially be very useful in future drug design of small-molecule compounds specifically targeting neuronal and cardiac excitability.

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Species referenced: Xenopus laevis
Genes referenced: kcna2 tbx2

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	Figure 1. The lipoelectric mechanism and binding sites for other compounds. (A) Schematic illustration of the PUFA effect on the Shaker channel: negatively charged PUFAs shift the voltage dependence of a Kv channel in a negative direction along the voltage axis. (B) A PUFA binds with its hydrophobic acyl tail in the hydrophobic lipid bilayer or a hydrophobic pocket in the channel. From this position, the negatively charged carboxyl group of the PUFA electrostatically attracts the positively charged voltage sensor to open the intracellular gate of the ion channel. (C) Side view of the Kv1.2/2.1 chimera with Shaker side chains. Back and front domains are removed for clarity. Note that the VSDs and pore domains shown are from different subunits. Residues critical for quaternary ammonium compounds (Zhou et al., 2001) (I470 and V474 in green), pore-blocking toxins (MacKinnon et al., 1990) (D431, T449, and V451 in magenta), voltage sensor–trapping toxins (Swartz and MacKinnon, 1997) (L327, A328, and V331 in red), and retigabine (Lange et al., 2009) (I400, G406, V407, M440, and A464 in yellow) are shown as sticks. The gating charges R362, R365, R368, and R371 are marked as blue sticks. Residue numbering refers to Shaker.
	Figure 2. Strategy to determine the PUFA action site. (A) Sequence of segment S3–S6 for the Shaker K channel. //, the extracellular linkers are omitted; *, tested residues. Underlined residues mark helical transmembrane segments. (B) Structures of the Shaker K channel in an open state (based on the Kv1.2/2.1 chimera; Long et al., 2007). View from the extracellular side (left). Only one VSD and part of the pore domain are shown. View from the membrane side (right) as indicated by the arrow in the left panel. Only one VSD and the closest pore domain from another subunit are shown. Selectivity filter regions from all four subunits are displayed in cyan. The blue residues are the four most extracellular gating charges in S4 (R362, R365, R368, and R371). Red residues are explored in the present investigation. (C) Only the negatively charged form of the carboxyl group affects the voltage sensor. The effect is pH dependent. The introduction of a fixed positive charge close to the PUFA changes the local pH, deprotonates the carboxyl group, and potentiates the PUFA effect on the voltage sensor. The closer the charge is to the PUFA, the larger the potentiation is.
	Figure 3. Effects of DHA and MTSEA+ at pH 7.4 on WT-IR and three mutations. Graphs display representative G-V curves with current traces as insets for voltages corresponding to 10% of Gmax in control solution. Eq. 2 is used for the fit. (A) Data for WT-IR. 70 µM DHA shifts the control curve for WT-IR with −4.7 mV (left). V1/2 = −42.8 and −47.5 mV, and s = 16.1 mV. MTSEA+ modification does not shift the control curve (middle). V1/2 = −38.9 and −38.9 mV, and s = 7.4 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −39.7 and −45.4 mV, and s = 8.2 mV. (B) Data for F416C. 70 µM DHA shifts the control curve for F416C with −2.8 mV (left). V1/2 = −45.7 and −48.5 mV, and s = 14.0 mV. MTSEA+ modification shifts the control curve with +28.2 mV (middle). V1/2 = −46.2 and −18.0 mV, and s = 14.0 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −18.0 and −21.8 mV, and s = 14.0 mV. (C) Data for I360C. 70 µM DHA shifts the control curve for I360C with −3.1 mV (left). V1/2 = −33.5 and −36.6 mV, and s = 8.7 mV. MTSEA+ modification shifts the control curve with +8.8 mV (middle). V1/2 = −34.7 and −25.9 mV, and s = 12.4 mV. The DHA-induced shift is increased to −8.5 mV after modification (right). V1/2 = −27.3 and −35.8 mV, and s = 16.1 mV. (D) Data for I325C. 70 µM DHA shifts the control curve for I325C with −2.9 mV (left). V1/2 = −40.1 and −43.0 mV, and s = 17.2 mV. MTSEA+ modification shifts the control curve with +16.9 (middle). V1/2 = −39.4 and −22.5 mV, and s = 13.7 mV. The DHA-induced shift is increased to −4.4 after modification (right). V1/2 = −22.6 and −27.0 mV, and s = 13.5 mV.
	Figure 4. Localization the PUFA action site. (A) Extracellular view of the Shaker K channel in an open state (based on the Kv1.2/2.1 chimera; Long et al., 2007). Only one VSD and part of the pore domain are shown. The blue residues are the four most extracellular gating charges (R362, R365, R368, and R371). The selectivity filter is shown in cyan. Green residues (I325, A359, and I360) have the largest impact on the PUFA-induced shift of the G-V curve, the yellow residue (T329) has a smaller but significant effect on the PUFA-induced shift, and red residues have no significant effects on the PUFA-induced shifts. The negative charge denotes an approximate position of the PUFA carboxyl charge affecting the voltage sensitivity of the Shaker K channel. (B) Side view of the channel with I325, A359, and I360 in green, T329 in yellow, and residues with no significant effects on the PUFA-induced shifts in red. Not investigated residues in S3 are shown in cyan, and those in S4 are shown in blue. All residues in space fill.
	Figure 5. Effect of DHA on the early activation steps versus the opening step. (A) A simple scheme for the ion channel kinetics. C0 to C4 denote closed states with 0–4 activated voltage sensors where the voltage sensors move independently of each other. O is the open state. (B) Theoretical G-V curves and Q-V curves for WT-IR and the ILT channel generated from the model in A. For calculations, see Eqs. 4–7 in Materials and methods. (C) 70 µM DHA at pH 9.0 increases the ion current at +60 mV in the ILT channel. Holding voltage is −80 mV. (D) The G-V curve is shifted −30 mV for the ILT channel. Eq. 2 is fitted to the experimental data as explained in Materials and methods. V1/2 = 118 mV for control and 88 mV for DHA. s = 22.6 mV and A = 0.271 mS in both curves. (E) Integrated OFF gating currents from the ILT/W434F mutation (n = 3). 70 µM DHA at pH 9.0 shifts the control curve −5 mV. (F and G) Calculated effects of DHA on open probability (F; G-V) and gating charge movement (G; Q-V) using Eqs. 4–7. Continuous lines are control curves. Dashed curves are DHA-affected curves. DHA was set to shift Vαβ with −5 mV and Vγδ with −30 mV for both channels. (H) Summary of DHA-induced shifts from both experiments and models. Data are expressed as mean ± SEM (n = 3–9).
	Figure 6. Strategy to test if PUFA facilitates horizontal S4 movement. (A) Schematic illustration of the assumed charge-transfer motion used for distance calculations (Eq. 3). The only change in electric charges between each gating state is that one gating charge is moved from the intracellular side to the extracellular side. Gating charges within the membrane electric field pair up with conserved negative charges. (B) Structure of the Shaker K channel in the open state (based on the Kv1.2/2.1 chimera). One VSD and part of the pore domain are shown. The four most extracellular-positive charges in S4 are colored: red, R362; orange, R365; green, R368; blue, R371. The negative charge denotes the position for the effective PUFA molecule. Arrows denote the distances from the effective PUFA site to the charge of R362 and to the site where the positive charges of R365 and R368 emerge on the surface, respectively. (C) Structural comparison of an arginine, a MTSES−-modified cysteine, and a MTSET+-modified cysteine.
	Figure 7. Effect of gating charge mutations on DHA sensitivity. (A) Data for WT-IR. Structure of the Shaker K channel in the open state (based on the Kv1.2/2.1 chimera), with residues R362, R365, R368, and R371 in blue (left). One VSD and part of the pore domain are shown. Current traces at −40 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −43.5 and −53.2 mV, and s = 11.3 mV. (B) Data for R362−. Residue R362 are shown in red, and residues R365, R368, and R371 are shown in blue (left). Current traces at −35 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −36.8 and −34.9 mV, and s = 25.9 mV. (C) Data for R362+. Residues R362, R365, R368, and R371 are shown in blue (left). Current traces at −45 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −45.9 and −58.3 mV, and s = 13.1 mV. (D) Data for A359+. Residues A359, R362, R365, R368, and R371 are shown in blue (left). Current traces at −5 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −9.5 and −33.0 mV, and s = 18.1 mV. (E) Summary of experimental data for gating charge mutants. Data are expressed as mean ± SEM (n = 4–9).
	Figure 8. Possible interpretations of differential effects on different steps. (A) Three possible models for the S4 movement during the opening step. (B) The new PUFA action site compared with previously described sites. The Shaker channel is viewed from the extracellular side. The color coding follows that from Fig. 1 C. Green denote residues critical for the binding of quaternary ammonium compounds, magenta is for pore-blocking toxins, red is for voltage sensor–trapping toxins, yellow is for retigabine, and orange is for PUFA in the present investigation. The gating charges R362, R365, R368, and R371 are marked as blue sticks.

References [+] :

Baker, Three transmembrane conformations and sequence-dependent displacement of the S4 domain in shaker K+ channel gating. 1998, Pubmed