A molecular framework for temperature-dependent gating of ion channels

Abstract

Perception of heat or cold in higher organisms is mediated by specialized ion channels whose gating is exquisitely sensitive to temperature. The physicochemical underpinnings of this temperature-sensitive gating have proven difficult to parse. Here, we took a bottom-up protein design approach and rationally engineered ion channels to activate in response to thermal stimuli. By varying amino acid polarities at sites undergoing state-dependent changes in solvation, we were able to systematically confer temperature sensitivity to a canonical voltage-gated ion channel. Our results imply that the specific heat capacity change during channel gating is a major determinant of thermosensitive gating. We also show that reduction of gating charges amplifies temperature sensitivity of designer channels, which accounts for low-voltage sensitivity in all known temperature-gated ion channels. These emerging principles suggest a plausible molecular mechanism for temperature-dependent gating that reconcile how ion channels with an overall conserved transmembrane architecture may exhibit a wide range of temperature-sensing phenotypes. :

Figure 1. Design template for engineering a temperature modulated ion channel

(A) Arbitrary relative open probability vs voltage (P_OV) curves for a heat-sensing channel (top) and a cold-sensing channel (bottom) at two temperatures (red: high temperature, blue: low temperature). Arrows indicate the shifts in the curve on heating. (B) ΔG vs T profiles simulated using the equation: ΔG(T) = ΔH_C + ΔC_P(T−T_C) − TΔC_Pln(T/T_C) for two processes, one with a positive ΔC_P (solid curve, ΔC_P = 3 kcal/(mol.K)) and the other with a negative ΔC_P (dotted curve, ΔC_P = −3 kcal/(mol.K)) (see also Fig. S1). In both cases. the critical temperature, T_C, was 308K (35°C), at which the change in enthalpy, ΔH_C, was −3 kcal/mol. The heat sensing and cold sensing regimes of the curves are indicated by the red and blue colors respectively. (C) Relative P_OV curve of the wild-type Shaker K_V channel at 28°C (red) and 8°C (blue) (measured from tail currents) (see also Fig. S1). (D) A model of a hydrated voltage-sensor, deduced from the crystal structure of the K_V1.2/2.1 paddle chimera (see Extended experimental procedures in Supplementary Material), showing occupancy of water within the crevices of the voltage-sensor and the sites that were perturbed in this study. The S4 helix is shown as a cartoon while the S1-S3 helices are shown as ribbons. (E) Fractional buried surface area of different residues in the transmembrane segments S1-S3 (residues numbered according the 2R9R structure). The dotted horizontal lines indicate the cut-off for buried surface areas. The bars are colored according to the polarity index of the residue position, deduced from an alignment of 360 K_V channel sequences. Residues targeted in this study are marked with arrows, with the residue numbers corresponding to Shaker K_V channel. The residue marked with an asterisk, while enriched in polar residues in the alignment, is a hydrophobic residue in Shaker K_V channel and was not studied here.

Figure 2. Crevice facing residues of S1-S3 segments sensitize channel opening to temperature

Relative open-probability vs voltage-curves for different perturbations at sites S240 (A), E293 (B) and Y323 (C) mutant, deduced from measurements of tail currents at 5mV voltage intervals (see also Fig. S2). Unless otherwise mentioned, in all cases blue curves indicate measurements at 8°C while red curves indicate measurements at 28°C. The mutation corresponding to each panel is listed in the top left corner of each panel. (D) Correlation of temperature dependent change in the median voltage of channel opening (ΔV_M) with the hydrophobicity of perturbation (H-vH scale of biological hydrophobicity (Hessa et al., 2005)) for sites S240 (left panel), E293 (middle panel), Y323 (right panel). The color gradient is used to show the transition from heat (negative ΔV_M) to cold sensitivity (positive ΔV_M) for the spectrum of mutations. (E) Correlation of median voltage of channel opening (V_M) at 28°C, with the hydrophobicity of perturbation for sites S240 (left panel), E293 (middle panel), Y323 (right panel)

Figure 3. Non-charged residues in S4 segment modulate temperature-sensitivity

(A) Relative P_OV curves, at two temperatures (28°C, red and 8°C, blue) for hextuplet mutations in the S4 segment where six residues are simultaneously mutated to Ile (left), Met (middle) or Ala (right). (B) Relative P_OV curves, at two temperatures (28°C, red and 8°C, blue) for quadruplet mutations in the S4 segment where four residues are simultaneously mutated to Ile (left), Met (middle) or Ala (right). (C) Correlation of temperature dependent change in the median voltage of channel opening (ΔV_M) with the hydrophobicity of perturbations for the Top (left panel), Middle (middle panel) and Bottom (right panel) doublet mutations (see also Figs. S3). The color gradient is used to show the transition from heat (negative ΔV_M) to cold sensitivity (positive ΔV_M) for the spectrum of mutations. (D) Correlation of median voltage of channel opening (V_M) at 28°C with the hydrophobicity of perturbations for the Top (left panel), Middle (middle panel) and Bottom (right panel) doublet mutations. (E) Relative P_OV curves at two temperatures (28°C, red and 8°C, blue) for doublet Ser mutations.

Figure 4. Enhancement of heat sensitivity by combining temperature-sensitive perturbations

(A) Relative P_OV curves for the mutant Y323I-S2Mid at 28°C and 8°C. Horizontal bar plot in the inset shows the temperature dependent shifts (ΔV_M) of the mutant (orange), compared with that of Y323I (red) and S2Mid (yellow). (B) Relative P_OV curves for the mutant Y323I-M6 at 28°C and 15°C. Horizontal bar plots in the inset show the shifts in the median voltage of activation (ΔV_M) for Y323I-M6 (orange), Y323I (red) and M6 (yellow). For the Y323I and M6 the shifts correspond to 20°C change in temperature while for the Y323I-M6 the orange bar corresponds to a 13°C change in temperature (the dotted bar corresponds to the shift of the mutant for a 20°C change in temperature expected from a linear extrapolation of the experimental result). (C) and (D) Semi-logarithmic plots of fractional outward current, at -20 mV, at different temperatures vs inverse of temperature, measured over 28°C to 8°C, at 1°C intervals, for S2Bot (C) and Y323I-M6 (D) compared with that of the wild-type channel. Shaded region indicates the 10° - 14°C temperature regime over which the Q₁₀ value was calculated. Using the formula: ΔH = RT²lnQ₁₀/10 described by Clapham and Miller (2011), the ΔH for the two mutants (at -20mV and room temperature, 20°C) are calculated to be 49.9 kcal/mol (for S2Bot, (C)) and 48.5 kcal/mol (for Y323I/M6 (D)).

Figure 5. Influence of voltage-sensing charges on temperature sensitivity

(A) Relative P_OV curves of R368N (triangles) and R368A (circles) at 28°C (red) and 8°C (blue) (B) Relative P_OV curves of R371Q (triangles) and R371A (circles) at 28°C (red) and 8°C (blue) (see also Fig. S4). (C)-(F) Relative P_OV curves of a temperature sensitizing mutant (Y323I, (C), (D); S2mid (E); Y323Q (F)) in the background of a charge neutralizing mutant (R368N, (D); R371Q, (D)-(F)). In each case the inset shows the temperature induced ΔV_M for the charge neutralizing mutant in red, the temperature sensitizing mutant in yellow and the combination mutant in orange. (G) ΔV_M for each of the temperature sensitizing mutants (Y323I, S2mid, Y323Q) in the background of a gating charge neutralization mutant (R368N (blue circle) or R371Q (red circles)) plotted against ΔV_M for each of the temperature sensitizing mutants (Y323I, S2mid, Y323Q) in the background of the native channel. The dotted line represents the regression line through the points, which has a slope of 2.06 ± 0.2. (H) Simulated V_M vs temperature profile for a cold sensitive process (ΔC_P > 0, top panel) and a heat sensitive process (ΔC_P < 0, bottom panel), deduced using the equation: V_M(T) = {ΔH_C + ΔC_P(T−T_C) − TΔC_Pln(T/T_C)}/z. In each case, dotted curves indicate the profiles for a process with low voltage-sensitivity and solid curves represent a process with high voltage-sensitivity. Arrows indicate the change in the change in V_M due to change in temperature.

Figure 6. State-dependent change in solvation is a possible mechanism of temperature dependent gating

The cartoons (Top) and (Bottom) depict the voltage-sensors in resting and activated conformations. Voltage-dependent change in the VSD is associated with a change in solvation of a residue. When the residue is polar (Top), activation will be conferred a negative ΔC_P (indicated by the blue halo around the hydration shell) and when the residue is non-polar (Bottom), activation will be conferred a positive ΔC_P (indicated by the red halo around the hydration shell). The opposite signs of ΔC_P in the two cases will lead to one of them (Top) being heat sensitive and the other (Bottom) being cold sensitive.