An external sodium ion binding site controls allosteric gating in TRPV1 channels
- National Institutes of Health, United States
Figures
Figure 1 with 4 supplements
Substitution of extracellular Na+ with NMDG+ increases TRPV1-mediated currents.
(A) Side view in ribbon representation of the transmembrane domains of two opposing TRPV1 subunits (as indicated by the black arrow, extracellular face on the top, intracellular face on the bottom) …
Figure 1—figure supplement 1
Substitution of external Na+ with NMDG+ induces channel rundown at room temperature with high cell-to-cell variability.
(A) Two representative TRPV1 current time courses obtained from a train of voltage ramps in the whole-cell configuration, constructed by plotting the mean currents at -120 (triangles) and +120 mV …
Figure 1—figure supplement 2
Activation of rat TRPV1 channel orthologues by substituting external Na+ with NMDG+.
Representative current families recorded from outside-out patches containing TRPV1 channels from different species (mouse, human and chicken) at room temperature. Currents were elicited by voltage …
Figure 1—figure supplement 3
Comparison of TRPV1 channel I-V relations measured using voltage steps and voltage ramps.
(A) Representative whole-cell TRPV1 current families obtained at room temperature in response to 100-ms voltage pulses from -120 to +140 mV in 10-mV increments and recorded in the absence and …
Figure 1—figure supplement 4
Theoretical I-V relations in the presence and absence of external Na+ obtained with the Goldman-Hodgkin-Katz current equation.
Superposition of the I-V relations obtained from voltage ramps (Figure 1C) and theoretical I-V curves calculated using the Goldman-Hodgkin-Katz current equation (red curves) with a permeability of …
Figure 2 with 1 supplement
Extracellular sodium ions are allosteric inhibitors of the TRPV1 channel.
(A, left) Representative recordings at +90 mV performed on outside-out patches containing a few TRPV1 channels in the presence of 130 mM external Na+ (top) or NMDG+ (bottom) at room temperature. The …
Figure 2—figure supplement 1
Monovalent cation selectivity of the external Na+-binding site in TRPV1.
(A) Voltage-ramps (darker thin curves are the mean, lighter-colored envelopes the SEM, n = 5) obtained in the whole-cell configuration in the presence of different extracellular cations (and 130 mM …
Figure 3 with 1 supplement
External Na+, H+ and DkTx modulate the TRPV1 channel through overlapping mechanisms involving E600 in the extracellular pore.
(A) Extracellular Na+ dose-response relations measured at different extracellular pH values at +120 mV in the whole-cell configuration in response to voltage ramps (mean ± SEM, n = 3–7). Experiments …
Figure 3—figure supplement 1
The location of E600, the extracellular pore turret and the binding site for DkTx within the outer pore of TRPV1, and the role of external Mg2+ ions in TRPV1 modulation.
(A) Side view of a ribbon representation of the transmembrane domain of the TRPV1 channel bound to DkTx/RTx (refined structural model for TRPV1 with the docked solution structure of DkTx) (Bae et …
Figure 4 with 2 supplements
Temperature-dependent gating of TRPV1 in the presence of external Na+.
(A) Representative whole-cell current family in the presence of external Na+ elicited by a train of pulses from -90 to +90 mV while increasing temperature (temperature vs time plot is shown on the …
Figure 4—figure supplement 1
Estimation of the temperature-dependence of ion conduction through an open channel for obtaining Po-T relations.
(A) Scheme of the custom-modified temperature-controlled microincubator stage (HCMIS and PTC-10 controller, Ala Scientific, Farmingdale, NY) used to obtain I-T relations. A round glass coverslip was …
Figure 4—figure supplement 2
Individual Po-T relations in the presence of external Na+ measured over a wide range of temperatures uncover the presence of multiple temperature-dependent components in the gating mechanism of the TRPV1 channel.
(A) Normalized I-T relation (mean ± SEM, grey circles) obtained from data in the presence of 130 mM external Na+ (Figure 4B) at +90 mV with superimposed I-T relations from individual cells (colored …
Figure 5 with 3 supplements
External Na+ has a strong influence on temperature-dependent gating of TRPV1.
(A) Representative whole-cell current family obtained as in Figure 4A in the absence of external Na+. The temperature vs time plot is shown on the right panel. Dotted lines denote the zero-current …
Figure 5—figure supplement 1
Temperature-dependent inactivation of TRPV1 channels in the absence of external Na+ can be partially reversed by capsaicin.
(A) Representative whole-cell current time course (lower panel) elicited by voltage steps from -90 to +90 mV while increasing temperature (time course for temperature is shown on the top panel) …
Figure 5—figure supplement 2
Scaling of Po-T relations based on estimates of absolute Po at room temperature from macroscopic I-V relations and noise-analysis.
(A) Mean normalized I-V relations at room temperature obtained in the whole-cell configuration using voltage ramps in the presence of extracellular solutions containing different Na+ concentrations …
Figure 5—figure supplement 3
Perfusion-mediated temperature control.
(A) Schematic illustration of the perfusion-based temperature-control system used for rapid temperature jumps and I-V relations at low temperatures. Solutions kept in elevated reservoirs (for …
Figure 6 with 1 supplement
The response of the TRPV1 channel to heating is dominated by distinct conformational transitions over different temperature ranges.
(A) Normalized Po-T relations (mean ± SEM, n = 3) obtained in the presence of 0 Nao and capsazepine (Cpz). Data in 0 Nao without capsazepine are shown for comparison. The essential features of the Po…
Figure 6—figure supplement 1
Po-T relations from individual cells obtained with different concentrations of external Na+.
(A) Mean Po-T relation at +90 mV in the absence of external Na+ (0 Nao, large yellow circles, Figure 5C, no-T relation predicted by model i (Figure 7A) in the absence of external Na+ using the …
Figure 7 with 5 supplements
An allosteric framework for TRPV1 channel gating.
(A) Scheme for Model i with two temperature-dependent transitions (horizontal arrows) given by equilibrium constants J1 and J2 of the form J(T) = exp(-(ΔHo-TΔSo)/RT)) and a temperature-independent …
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Figure 7—source data 1
Analytical expressions for Po in different models.
- https://doi.org/10.7554/eLife.13356.022
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Figure 7—source data 2
Parameters for the mathematical gating models.
- https://doi.org/10.7554/eLife.13356.023
Figure 7—figure supplement 1
Alternative models for describing temperature-dependent gating of TRPV1 and its modulation by external Na+.
(A) Scheme for Model ii with individual conformational states represented as squares. ‘C’ labels denote closed states, ‘O’ open states and the subscript ‘Na’ denotes Na+-bound states. In the absence …
Figure 7—figure supplement 2
High concentrations of external Na+ do not fully prevent TRPV1 channel activation by positive voltages, capsaicin or heat.
(A) Normalized I-V relations obtained from voltage ramps measured in the whole-cell configuration with an intracellular solution containing 600 mM NaCl and the following extracellular solutions (in …
Figure 7—figure supplement 3
Assessing the influence of voltage on temperature-dependent gating of TRPV1 channels in the absence of external Na+.
(A) Representative whole-cell TRPV1 current families obtained at 8°C in response to 400 ms voltage pulses from -120 to +140 mV in 10 mV increments (only traces for steps to even voltages are shown …
Figure 7—figure supplement 4
The effect of a change in heat capacity associated with the operation of the temperature-sensor on the predictions of the allosteric gating model.
(A) Graph showing how J1(T) (blue) and J2(T) (red) (see model i in Figure 7A) change as a function of temperature when they are associated with a change in heat capacity (dashed curves, parameters …
Figure 7—figure supplement 5
Incorporating temperature-dependent inactivation into the allosteric model.
(A) Theoretical plot of temperature vs time used for introducing temperature-dependent inactivation into the predictions of model i (see Materials and methods). (B) Theoretical Po-T relations (model …
Figure 8 with 1 supplement
The effect of capsaicin on temperature-dependent gating of TRPV1.
(A) Po-T relations (mean ± SEM, n = 4–14) obtained in the presence of 130 mM external Na+ and different concentrations of capsaicin (see Figure 8—figure supplement 1A for Po-T relations from …
Figure 8—figure supplement 1
Po-T relations from individual cells for different concentrations of capsaicin and predictions of the allosteric model over an extended range of temperatures.
(A) Theoretical Po-T relations (thick continuous curves, model i with capsaicin – Figure 8B, Po-equation in Figure 7—source data 1A, parameters in Figure 7—source data 2A) calculated for three …
Figure 9 with 1 supplement
Deletion of the outer pore turret uncovers temperature-sensitive gating of TRPV1 in saturating capsaicin.
(A) Three representative TRPV1 Δ604–626 whole-cell I-T relations measured in 130 mM external Na+ in response to pulses from -90 to +90 mV as in Figure 4A and B. The dotted line is the zero-current …
Figure 9—figure supplement 1
Influence of the pore turret of TRPV1 on temperature-dependent gating and modulation by sodium.
(A) External Na+ dose-response relations for TRPV1 Δ604–626 (open circles, mean ± SEM, n = 4) measured at +90 mV from voltage-ramps in the whole-cell configuration. Closed circles are data for WT …
Figure 10 with 1 supplement
The binding of DkTx to the outer pore of TRPV1 effectively ablates temperature-dependent gating over a wide range of temperatures.
Po-T relation (mean ± SEM, n = 9) for WT TRPV1 obtained in the presence of external Na+ and DkTx. The dotted line denotes the mean Po at 22°C and +90 mV as estimated from the I-V relations in DkTx …
Figure 10—figure supplement 1
DkTx binding to TRPV1.
(A) Overlay of the side views of the S3-S6 segments of one TRPV1 subunit and the S5-S6 segments of an adjacent subunit in the apo (subunit 1, S3-S4 in light pink, S5-S6 in magenta; subunit 2, S5-S6 …
