Gating of TRP channels: a voltage connection?

Abstract

TRP channels represent the main pathways for cation influx in non-excitable cells. Although TRP channels were for a long time considered to be voltage independent, several TRP channels now appear to be weakly voltage dependent with an activation curve extending mainly into the non-physiological positive voltage range. In connection with this voltage dependence, there is now abundant evidence that physical stimuli, such as temperature (TRPV1, TRPM8, TRPV3), or the binding of various ligands (TRPV1, TRPV3, TRPM8, TRPM4), shift this voltage dependence towards physiologically relevant potentials, a mechanism that may represent the main functional hallmark of these TRP channels. This review discusses some features of voltage-dependent gating of TRPV1, TRPM4 and TRPM8. A thermodynamic principle is elaborated, which predicts that the small gating charge of TRP channels is a crucial factor for the large voltage shifts induced by various stimuli. Some structural considerations will be given indicating that, although the voltage sensor is not yet known, the C-terminus may substantially change the voltage dependence of these channels.

Figure 1. Voltage dependence of TRPs (TRPM4)

A, activation curve of a Shaker K⁺ channel. Because of the large gating charge, activation occurs in a very narrow voltage range. B, activation curve of a voltage-dependent TRP channel. This curve is shallow, which reflects a small gating charge. The open probability changes over a broad voltage range, which for most TRP channels extends into a range of non-physiological positive potentials. Activation of TRP channels in response to various stimuli results from a shift of their voltage dependence towards negative potentials and opening at physiological potentials. C, activation of TRPM4 channels (inside out patch, 300 μm Ca²⁺ at the inner side of the membrane) by depolarizing voltage steps. Note deactivation at negative potentials and activation at positive potentials. Patches were held at 0 mV, steps from −100 mV to +180 mV (increment 20 mV) for 400 ms, and then back to −100 mV for 100 ms. D, current–voltage relationships (I–V curves) from the experiment shown in C: steady state I–V curves (○) show outward rectification, I–V curves from the tail currents saturate at positive prepotentials (▵). E, voltage dependence of the time constants of current activation obtained from mono-exponential fits. F, voltage dependence of the open probability, P_o, of the TRPM4 channel obtained from tail current measurements as shown in C and D. Continuous line is fit with the Boltzmann equation, V_½= 92 mV a s = 32 mV, i.e. z = 0.75. G, calculation of the rate coefficients α and β for the two state model from E and F. Data were fitted to y = y_o + Aexp(t/τ) (continuous lines).

Figure 2. Examples of voltage shifts of activations curves of different TRP channels by various gating modifiers

A, voltage dependence of the open probability of TRPV1 channels at 17°C (▵) and 42°C (○). The inset shows the respective current families for a voltage step programme (holding potential 0 mV, test steps from −120 to +160 mV, increment 40 mV), back step to +60 mV. Note the dramatic shift towards negative potentials from +198 to −33 mV. B, same protocol as in A, but for TRPM8 at 37°C (▵) and 15°C (○). Note the leftward shift of the activation curve. V_½ changed from +203 to 53 mV. C, same voltage protocol as in A. TRPV1 is activated by 100 nm capsaicin (○) causing a leftward voltage shift of from +95 to −22 mV. Temperature was 24°C. D, 30 μm menthol (○) induced a leftward shift from 203 to 74 mV of the TRPM8 activation curve at 34°C (circles are controls, same protocol as in A). E, TRPM4 was activated in inside-out patches by different [Ca²⁺]_i. Voltage protocol consists of steps from −100 to +160 mV (increment 40 mV) from a holding potential of 0 mV. A change from 10 μm [Ca²⁺]_i (▵) to 1000 μm [Ca²⁺]_i (○) induced a leftward shift of the activation curve from 162 to 68 mV. F, TRPM4 activation in the presence of 300 μm [Ca²⁺]_i. Neutralizing the positive charge K919 caused a shift of V_½ from +60 (wild type TRPM4) to +192 mV (K919A mutant). Holding potential was 0 mV, steps from −25 to 250 (mV). (Calibration bars in the insets are identical for examples shown in each panel). (A–D are adapted from Voets et al. (2004) with permission from Nature, http://www.nature.com; E is adapted from Nilius et al. (2004).)

Figure 3. A thermodynamic analysis of the temperature dependence of TRPM8 and TRPV1

A, Arrhenius plots for the rate constants α and β of TRPM8 for the two state model at −80 (Δ), +80 (▪) and +160 mV (○). The slope corresponds to the enthalpy. The enthalpy for channel opening is small, corresponding to a Q₁₀ values of 1.2, but is large for channel closing (corresponding Q₁₀ is 9.4). From the shifts in V_½, the following data were obtained: ΔH_o = 13 kJ mol⁻¹, ΔH_c = 170 kJ mol⁻¹, ΔS_o = −166 J mol⁻¹ K⁻¹, ΔS_c = 392 J mol⁻¹ K⁻¹. B, same analysis as in A but for TRPV1. Note that the enthalpy for channel opening is much higher (corresponding Q₁₀ is 14.8) than for channel closing (Q₁₀ is 1.35). Considering the shifts in V_½, the following data were obtained: ΔH_o = 205 kJ mol⁻¹, ΔH_c = 21 kJ mol⁻¹, ΔS_o = 468 J mol⁻¹ K⁻¹, ΔS_c = −130 J mol⁻¹ K⁻¹. C, shift of the potential for half-maximal activation of TRPM8 as a function of temperature. With V_½ = (ΔH – ΔST)/zF, the positive slope corresponds to the negative change in entropy. Menthol induced a parallel shift of this curve toward more negative potentials, likely indicating a change in enthalpy. (A and B are adapted from Voets et al. (2004) with permission from Nature, http://www.nature.com.)

Figure 4. Gating modification of TRPM4

A, a structural model of the C-terminus of TRPM4 from S1044 to D1214 (see text for more details), viewed using DS ViewerPro 5.0 software (Accelrys Inc., San Diego, CA, USA) (parental structure score 1.29, serine acetyltransferase-apoenzyme, chain A (1smmA) score). Indicated are the two putative Ca²⁺–calmodulin binding sites in blue (for details see Nilius et al. 2005). α-Helices are shown in red, turns in grey. The putative binding site for decavanadate, R¹¹³⁶ARDKR¹¹⁴¹) is shown in cyan (for details see Nilius et al. 2004). In the modelled coiled-coil region (1134–1154 and 1158–1185, centre of the figure) are two PKC phosphorylation sites indicated in yellow (Nilius et al. 2005). B, deletion of the calmodulin binding site induced a rightward shift of the activation curve of TRPM4. The inset shows current traces from voltage steps for controls (○) and the deletion mutant of the first C-terminal Ca²⁺–calmodulin binding site ΔA1076–S1098 (▵). [Ca²⁺]_i was 100 μm, voltage steps from −100 to +200 mV, 20 mV increment for the control and from −100 to +260 mV, increment +40 mV for the deletion mutant (inside out patches). Note the shift of V_½ from +112 to +235 mV. C, effects of decavanadate (DV) on the activation curve of TRPM4. The inset shows current traces from inside patches with [Ca²⁺]_i of 300 μm (controls, ▵ and after application of 10 μm DV, ○). Activation curves show a shift of more than 200 mV toward negative potentials in the presence of DV. Note the very shallow voltage dependence, and the loss of activation and deactivation kinetics (Nilius et al. 2004). D, mutation of the C-terminal putative PKC phosphorylation sites S1145 and S1152 to aspartates induces a leftward shift of the TRPM4 activation curves. The inset shows current traces from inside out patches with wild-type TRPM4 (▵) and with the mutation S1145D (○). Shift is from V_½ of +124 to +12 mV (100 μm [Ca²⁺]_i, steps from −75 to +200, +150 mV, respectively, holding potential 0 mV). This shift is in agreement with a sensitizing effect of PKC phosphorylation of TRPM4 (Nilius et al. 2005). (C, modified from Nilius et al. (2004).).