Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pH(i) with pH(o) 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pH(i) less than or equal to 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, g(H)-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ cur rent amplitude was estimated from stationary or quasi-stationary variance, sigma(H)(2). We analyze sigma(H)(2) data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pH(i) 6.5, and increased nearly fourfold to 140 IS at pH(i) 5.5, but was independent of pH(o). In contrast, the macroscopic g(H) was only 1.8-fold larger at pH(i) 5.5 than at pH(i) 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pH(i) 6.5 and 0.95 at pH(i) 5.5. Because the unitary conductance increases at lower pH(i) more than the macroscopic g(H), the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pH(i) less than or equal to 5.5 near V-threshold (the voltage at which g(H) turns on), single channel-like current events were observed with amplitudes 7-16 fA.
