Inhibition of human acid-sensing ion channel 1b by zinc

Abstract

Acid-sensing ion channel 1b (ASIC1b) is expressed in peripheral sensory neurons and has been implicated in nociception. Understanding the modulation of ASIC1b will provide important insight into how ASIC1b contributes to pain sensation. In our previous study, we showed that zinc, an important modulator of pain sensation, reduces rat ASIC1b current. However, rat ASIC1b shows several important differences from its recently identified human homolog. Most noticeably, human ASIC1b (hASIC1b) has a sustained component, which may play a role in persistent pain. Therefore, we tested here the hypothesis that zinc modulates the current properties of hASIC1b. Bath application of zinc suppressed the peak amplitude of hASIC1b currents, with a half-maximum inhibitory concentration of 37 μM. However, zinc did not affect the sustained component of hASIC1b currents. The effect of zinc was independent of pH-dependent activation, steady-state desensitization, and extracellular Ca(2+), suggesting noncompetitive mechanisms. Further, we found that extracellular site(s) of the hASIC1b subunit is important for the effect of zinc. Mutating cysteine 196, but not cysteine 309, in the extracellular domain of the hASIC1b abolished the zinc inhibition. These results suggest that, through modulating cysteine196, zinc may have a modulatory role in acute pain.

Keywords: Acid-sensing ion channels; hASIC1b; pain; patch-clamp; zinc.

Figure 1

Co-application of zinc doesn’t affect hASIC1b currents in hASIC1b transfected CHO cells. A, Representative traces showing pH-6.0 elicited hASIC1b currents with co-application of different concentrations of zinc in CHO cells. Transient inward hASIC1b currents in CHO cells were recorded with drops in pH from 7.4 to 6.0 in whole-cell configurations at –60 mV. The duration of co-application with pH drops is 7 seconds. B & C, Quantification of relative peak current amplitude (B), and sustained-component (C) of pH 6.0-activated currents with co-application of zinc at different concentrations. Each point represents the average responses of five to eight cells. There are no significant differences between each group (p > 0.05, ANOVA). CTRL represents control.

Figure 2

Bath application of zinc suppresses peak amplitude, but not sustained component of hASIC1b currents concentration-dependently in hASIC1b transfected CHO cells. A, Representative traces showing the concentration-dependent blockade of hASIC1b currents triggered by dropping the pH from 7.4 to 6.0 in the presence of different concentrations of zinc. B, Concentration-inhibition curve of pH 6.0-induced currents by bath application of zinc. The IC₅₀ of zinc blockade is 36.45 ± 1.52 μM. Each point represents the average responses of eight cells. C, Quantification of relative sustained-component of pH 6.0-activated currents with bath application zinc at different concentrations. Each point represents the average responses of eight cells. There are no significant differences between each group (p > 0.05, ANOVA). CTRL represents control.

Figure 3

Zinc inhibition of hASIC1b currents in hASIC1b transfected CHO cells is independent of pH activation and steady-state desensitization. A, Original current traces showing inhibitory effects of 50 μM zinc with pretreatment on the pH-dependent activation of hASIC1b currents in CHO cells. The starting pH for all experiments was 7.4, the activating pH is indicated by bars above the trace and CHO cells were clamped at –60 mV. B, Quantification of relative peak amplitude of hASIC1b current inhibition by bath application of 50 μM zinc. Each point represents the average response of six to nine cells. There are no significant differences between different groups in the presence of 50 μM zinc (p > 0.05, ANOVA). C, Original current traces showing inhibitory effects of 50 μM zinc with pretreatment on steady-state desensitization of ASIC1b currents in CHO cells. Steady-state desensitization was induced by various conditioning pH values between 7.4 and 6.8 for ~6 min before application of pH 5.0. CHO cells were clamped at –60 mV. D, Quantification of relative peak amplitude of hASIC1b current inhibition on steady-state desensitization by pre-applied 50 μM zinc at various conditioning pH values (between 7.8 to 6.8). hASIC1b current was evoked by application of pH 5.0. Each point represents the average response of five to nine cells. There are no significant differences between each group in the presence of 50 μM zinc (p > 0.05, ANOVA). CTRL represents control.

Figure 4

Zinc inhibition of hASIC1b currents in hASIC1b transfected CHO cells is independent of calcium. A, Original current traces showing inhibitory effect of pre-applied zinc on hASIC1b currents in the presence of 2, 5 and 10 mM Ca²⁺. hASIC currents were activated by a drop in pH from 7.4 to 6.0 at a membrane potential of -60 mV. B, Quantification of relative peak amplitude of zinc inhibition at different concentrations of Ca²⁺. Each point represents the average response of five cells. There are no significant differences between different groups (p > 0.05, ANOVA). CTRL represents control.

Figure 5

Extracellular zinc is responsible for inhibition of the hASIC1b current in hASIC1b transfected CHO cells. A, Representative traces showing that inhibition of hASIC1b currents by bath application of 50 μM zinc (extracellular zinc) was not affected by inclusion of 50 μM zinc in the pipette solution (i.e. intracellular zinc). hASIC1b currents were activated by a drop in pH from 7.4 to 6.0. B, Quantification of relative peak amplitude of zinc inhibition in the presence of 30 μM zinc in the pipette. Each point represents the average response of six cells. C, Original traces showing that inhibition of hASIC1b currents by bath application of 50 μM zinc (extracellular zinc) under no added zinc in the pipette. hASIC1b currents were triggered by a drop in pH from 7.4 to 6.0. D, Quantification of relative peak amplitude of zinc inhibition under no added zinc in the pipette. Each point represents the average response of five cells. Asterisk indicates values significantly different from the control, t-test, **p < 0.01. CTRL represents control.

Figure 6

hASIC1b-C196A, but not hASIC1b-C309 mutant abolished the zinc inhibition. A, Representative traces showing the concentration-dependent blockade of hASIC1b-C309A mutant currents triggered by dropping the pH from 7.4 to 6.0 in the presence of different concentrations of zinc. B, Representative traces showing that hASIC1b-C196A mutation abolished the zinc inhibition in the presence of different concentrations of zinc with bath application. C, Concentration-inhibition curve of pH 6.0-induced currents on hASIC1b-C309A mutant by bath application of zinc. The IC₅₀ of zinc blockade is 31.4 ± 1.3 μM. Each point represents the average responses of five cells. D, Quantification of relative amplitude of pH 6.0-activated currents on hASIC1b-C196A mutant with bath application of zinc at different concentrations. Each point represents the average responses of six to eight cells. There are no significant differences among all groups p > 0.05, ANOVA). CTRL represents control.