Thermodynamics of heat activation of single capsaicin ion channels VR1

Abstract

Temperature affects functions of all ion channels, but few of them can be gated directly. The vanilloid receptor VR1 provides one exception. As a pain receptor, it is activated by heat >42 degrees C in addition to other noxious stimuli, e.g. acids and vanilloids. Although it is understood how ligand- and voltage-gated channels might detect their stimuli, little is known on how heat could be sensed and activate a channel. In this study, we characterized the heat-induced single-channel activity of VR1, in an attempt to localize the temperature-dependent components involved in the activation of the channel. At <42 degrees C, openings were few and brief. Raising the ambient temperature rapidly increased the frequency of openings. Despite the large temperature coefficient of the apparent activity (Q(10) approximately 27), the unitary current, the open dwell-times, and the intraburst closures were all only weakly temperature dependent (Q(10) < 2). Instead, heat had a localized effect on the reduction of long closures between bursts (Q(10) approximately 7) and the elongation of burst durations (Q(10) approximately 32). Both membrane lipids and solution ionic strength affected the temperature threshold of the activation, but neither diminished the response. The thermodynamic basis of heat activation is discussed, to elucidate what makes a thermal-sensitive channel unique.

FIGURE 1

Heat-evoked single-channel activity in oocytes expressing VR1. The temperature was elevated from 25 to 50°C, activating the channel at ∼42°C. Both channel opening and unitary current amplitude increased with temperature. Data were acquired from an inside-out patch at a holding potential of −60 mV, and low-pass filtered at 1 kHz for display.

FIGURE 2

Temperature dependence of single-channel P_o and unitary current amplitude of VR1 from inside-out patches. (A) The single-channel P_o exhibited a sigmoidal increase with temperature. The response initiated at ∼42°C with a half-activation threshold at 45.6 ± 0.4°C, and appeared to saturate after 50°C. (B) The unitary current amplitude was temperature dependent also, albeit to a lesser degree. (C) The van't Hoff plot of the equilibrium constant, K_eq, determined from the open channel probability (P_o). The plot could be approximately regressed by a linear line, corresponding to an enthalpy ΔH = 150 ± 13 kcal/mol and entropy ΔS = 470 ± 40 cal/mol · K, respectively. The equation used for fitting is described in the text. (D) The van't Hoff plot of the unitary current of VR1. The plot was approximately linear in the lower temperature ranges (41–45°C; right part of the curve) but became saturated at high temperatures (>45°C; left part). A linear regression of the values at the low temperature range (41–45°C) resulted in an activation energy of ΔH = 33 ± 4 kcal/mol. (E) Expected temperature dependence of whole-cell currents as determined from the unitary current amplitude and single-channel P_o. Similar to single-channel P_o, the product activates at ∼42°C and saturates at >50°C. In contrast, however, it is more strongly dependent on temperature, with a Q₁₀ = 40.5 ± 14.4. Recordings were made at the holding potential of −60 mV. Each point represents 11 experiments for P_o and the unitary current amplitude.

FIGURE 3

Temperature dependence of gap and burst durations. (A) The mean gap duration showed a significant temperature dependence, with Q₁₀ ≈ 7 at 40°C. Increasing temperature reduced the gap duration. Assuming the gap duration reflects an opening rate of the channel, a linear regression of the Arrhenius plot yielded an activation enthalpy of ΔH ≈ 39.5 kcal/mol and an entropy of 100 cal/mol · K, respectively. (B) Distributions of the individual gap durations at two temperature ranges. Superimposed on the histograms are the best exponential fits, where the dotted lines represent the individual components and the solid lines correspond to the sums. A best fitting required at least two exponentials. The time constants of the fits were ∼80 and 524 ms for 40–42°C, and 45 and 303 ms for 44–48°C, respectively. (C) Temperature dependence of burst durations. Increasing temperature prolonged bursts. The bursts had a temperature dependence Q₁₀ ≈ 32. The line through the data points represents an Arrhenius fit of the durations, assuming that they are determined by a closing rate of the channel. The fit gave a deactivation enthalpy of ΔH ≈ −70 kcal/mol and an entropy of ΔS ≈ −123 cal/mol · K. (D) Distributions of individual burst durations. Fitting of the distributions resolved four exponential components with time constants (ms): 0.5, 10, 83, and 350 for 40–42°C, and 0.3, 69, 458, and 2922 for 44–48°C, respectively. The individual components are shown as the dotted lines whereas the sums are shown as the solid lines.

FIGURE 4

Dwell-time histograms for the experiment shown in Fig. 1. The data were analyzed at 10 kHz and idealized using the segmental k-means method. Only the intraburst activity was analyzed. The long closures with durations >50 ms were excluded. The resultant dwell-time sequences were subject to a dead-time of 40 μs. Superimposed with the histograms are the distributions predicted from the results of maximum likelihood fitting. The dotted lines show the contributions of individual components. For this particular experiment, 2–6 closed and 2–5 open states were necessary for an adequate fit across all temperatures. The time constants and proportions of the components are listed in Table 1.

FIGURE 5

Temperature dependence of dwell-time components. (A) Time constants and relative proportions of the closed and open components plotted against temperature. Results were averaged from 11 experiments. The numbers of the resolved closed and open components were not all consistent across experiments and temperatures. However, they generally fell into three categories for both closures and openings, as evident from their time constant distributions. As such, they were grouped into three ranges: 0–0.4, 0.4–5, and >5 ms for the closures; and 0–0.4, 0.4–1, and >1 ms for the openings, which were named short (S), medium (M), and long (L), respectively. Degenerate components that belonged to the same group within an experiment were averaged with their relative proportions as the weighting factor. The resultant components exhibited a weak temperature dependence on their time constants. Increasing temperature had a more profound effect on their relative proportions. The solid lines through the data points on the time constant plots correspond to the best linear regressions. (B) Coupling between closed and open components, as determined from the relative volumes of the 2D exponentials in the 2D dwell-time distributions. The most significant coupling occurred between short closures and long openings. Increasing temperature further enhanced their occurrences. The couplings between other types of components were significant at low to medium temperature ranges, but tended to vanish at high temperatures.

FIGURE 5

Temperature dependence of dwell-time components. (A) Time constants and relative proportions of the closed and open components plotted against temperature. Results were averaged from 11 experiments. The numbers of the resolved closed and open components were not all consistent across experiments and temperatures. However, they generally fell into three categories for both closures and openings, as evident from their time constant distributions. As such, they were grouped into three ranges: 0–0.4, 0.4–5, and >5 ms for the closures; and 0–0.4, 0.4–1, and >1 ms for the openings, which were named short (S), medium (M), and long (L), respectively. Degenerate components that belonged to the same group within an experiment were averaged with their relative proportions as the weighting factor. The resultant components exhibited a weak temperature dependence on their time constants. Increasing temperature had a more profound effect on their relative proportions. The solid lines through the data points on the time constant plots correspond to the best linear regressions. (B) Coupling between closed and open components, as determined from the relative volumes of the 2D exponentials in the 2D dwell-time distributions. The most significant coupling occurred between short closures and long openings. Increasing temperature further enhanced their occurrences. The couplings between other types of components were significant at low to medium temperature ranges, but tended to vanish at high temperatures.

FIGURE 6

Effect of cholesterol enrichment and depletion on the heat response of VR1. The currents were recorded from VR1-expressing HEK 293 cells in whole-cell configuration using a ramp protocol stimulus (25–50°C in 40 s). The instantaneous currents at different temperatures were plotted. (A) Cells at the normal condition without any treatment exhibited an averaged response with the 10% and the half-activation at ∼43 and 47°C, respectively. (B) Incubation of cells in a high cholesterol solution right-shifted the activation temperature by 3–4°C. The depletion of cellular cholesterol with β-methyl cyclodextrin, either at 50 μM (C) or 2.5/5 mM (D), resulted in little change in the heat response. The half-activation of the currents remained at 47–48°C. Each trace in the figure represents a recording from a single cell.

FIGURE 7

Effect of ionic strength on the heat response of VR1. Three conditions are shown. (A) High concentration (200 mM Cs⁺) in pipette. (B) Low concentration (25 mM Cs⁺) in pipette. (C) Low concentration (25 mM Na⁺) in bath. Increasing the intracellular ionic strength had little effect on the activation temperature of the channel, with the 10% activation at 43–44°C and the half-activation at 47–48°C, both of which were similar to those at the control condition (140 mM Cs⁺ in pipette and 140 mM Na⁺ in bath). In contrast, lowering the intracellular ionic strength increased the activation temperature by ∼4°C, with the 10% activation shifted to 46–47°C and the half-activation to 51°C. Lowering the extracellular ionic strength, on the other hand, yielded the opposite effect. The activation temperature was reduced by ∼4°C, with 10% activation occurring at ∼37°C and half-activation at ∼44°C. The protocol of the stimulus used in these experiments was slightly different from that in the cholesterol experiments, consisting of a heating to the maximal temperature first, followed by a cooling to the room temperature. The heating followed a ramp protocol as described. The currents plotted were the instantaneous recordings. Only the currents during the heating phase are shown.