The effects of pH on the interaction between capsaicin and the vanilloid receptor in rat dorsal root ganglia neurons

Abstract

1. The vanilloid receptor of sensory neurons is a polymodal nociceptor sensitive to capsaicin, protons, heat and anandamide. Although it is known that interaction occurs between these different mediators the mechanism by which this occurs is poorly understood. In this study capsaicin elicited currents were recorded from vanilloid receptors found in adult rat isolated dorsal root ganglia (DRG) neurons under conditions of varying pH and the mechanism whereby protons can modulate this capsaicin response investigated. 2. Under whole-cell voltage clamp, modulating extracellular pH shifted the position of the capsaicin log(concentration)-response curve. Acidification from pH 9.0 to pH 5.5 lowered the EC50 values from 1150+/-250 nM to 5+/-2 nM with coincident change in the mean apparent slope factor from 2.3+/-0.3 to 0.9+/-0.2 and no change in maximal response. 3. The magnitude of the potentiation seen on reducing extracellular pH was not significantly affected by changes in extracellular calcium and magnesium concentration. 4. The response to capsaicin was not potentiated by a reduction in intracellular pH suggesting a site of action more accessible from the extracellular than the intracellular side of the membrane. 5. Potentiation by low pH was voltage independent indicating a site of action outside the membrane electric field. 6. At the single channel level, reducing extracellular pH increased channel open probability but had no significant effect on single channel conductance or open time. 7. These results are consistent with a model in which, on reducing extracellular pH, the vanilloid receptor in rat DRG neurons, changes from a state with low affinity for capsaicin to one with high affinity, coincident with a loss of cooperativity. This effect, presumed to be proton mediated, appears to involve one or more sites with pK(a) value 7.4-7.9, outside the membrane electrical field on an extracellularly exposed region of the receptor protein.

Figure 1

Potentiation of the response to capsaicin by low pH. DRG neuron was voltage clamped at −60 mV and capsaicin applied at pH 7.4 (single line) or pH 6.0 (double line) at either 10 nM (b, d) or 10 μM (c) as indicated. (a) shows response to pH 6.0 solution without capsaicin. Inward current downwards, responses to low pH alone not subtracted.

Figure 2

Effect of extracellular pH on the capsaicin log(concentration)-response curve. (a) Log(concentration)-response curves for capsaicin recorded at +60 mV with extracellular pH of 5.5 (circles), 7.4 (open diamonds) and 9.0 (triangles). Curve fitted of form [I_min−I_max/(1+(x/x₀)^p]+I_max (where; I_min is the minimum current, I_max the maximum, x₀ the x value for half maximal response and p the apparent slope factor). Current was normalized to that elicited by 10 μM capsaicin for each neuron. (b) Concentration of capsaicin for half maximal response at each of these pH values with additional such curves as a function of the extracellular pH. Curve fitted based on model described in text constrained to a lower limit of 4.5±1.7 nM calculated from the mean data at pH 5.5 and pH 6.0 (n=18). Upper limit of 1270±160 nM and pK_a value of 7.9±0.2 determined from best fit (n=75). (c) Slope factors from the curves in (a) and additional such curves as a function of extracellular pH. Curve of same form as in (b) with limits of 1.1±0.2 and 2.3±0.2 and pK_a value of 7.4±0.4, determined from best fit (n=75).

Figure 3

Effect of extracellular pH on the maximal capsaicin current. (a) Mean peak currents elicited by 10 μM capsaicin from the data in Figure 2 as a function of pH. (b) Effect of extracellular pH on the current elicited by 100 μM capsaicin in a single neuron at +40 mV. One hundred μM capsaicin was applied at pH 6.5 then in the maintained presence of capsaicin extracellular pH increased to pH 9, then returned to pH 6.5 as shown. (pH order was reversed in half the neurons tested). Only one set of recordings was used from each neuron corresponding to the first application of capsaicin to that dish and only one neuron was used from each dish. Decreasing the extracellular pH had very little effect on the capsaicin elicited current. (c) Mean data from experiments such as that in (b) with any current elicited by the pH change alone subtracted, showing the relative size of the current at pH 6.5 if that at pH 9.0 is 1 (n=8).

Figure 4

Effect of divalent cations on the potentiation of the capsaicin response. (a) Mean current elicited at −60 mV on application of 10 nM capsaicin at pH 7.4 (filled bars) or pH 6.0 (open bars) in standard or divalent solution as indicated. Currents elicited by pH alone were subtracted. (b) Current at pH 6.0/current at pH 7.4 for each neuron in the two solutions (n=11 and 8 respectively).

Figure 5

Effect of intracellular pH on the response to capsaicin. Current elicited by neurons held at −60 mV with internal pH 7.4 (buffered with either 10 or 40 mM HEPES) or pH 6.0 (buffered with 40 mM MES) to external application of 10 nM capsaicin at pH 7.4 (filled bars) or pH 6.0 (open bars) (n=8,9,9). Series resistance ⩽8 MΩ, ⩾5 min equilibration period after reaching whole-cell configuration before recording.

Figure 6

Voltage dependency of the potentiation of the capsaicin response by extracellular protons. (a) Current-voltage relationships at extracellular pH 7.4 (diamonds) and 7.0 (triangles) with subthreshold concentration of capsaicin, either 50 or 100 nM for each neuron. Current normalized to that at +80 mV and pH 7.4 for each neuron. Responses to application of pH in the absence of capsaicin subtracted (n=7). (b) Relative potentiation (current pH 7.0/current pH 7.4) as a function of voltage, normalized to that at −100 mV for each neuron (n=7).

Figure 7

Effects of pH on the response to capsaicin at the single channel level. (a) Current elicited by outside-out patch at +80 mV on application of 10 μM capsaicin at pH 8.5, pH 7.4 and pH 5.5 as indicated. Filtered at 2 kHz. (b) Same patch as in (a) showing single channel activity at a range of potentials. No activity was seen in this patch on application of the pH solutions alone. (c) Mean single channel currrent-voltage relationship, combined data from five patches at pH 7.4 (open diamonds) and reduced pH (filled triangles).

Figure 8

The effect of extracellular pH on mean channel open times. (a) Mean open time distributions of single channel activity in an outside-out patch at +80 mV with extracellular pH 8.5 (upper) and pH 7.4 (lower) each fitted with two exponentials. Inserts show data on the same scale to allow easier comparison of the relative frequencies. (b) Mean values for slower of these exponentials from 6 – 9 patches as a function of pH.

Scheme 1