Calcium-dependent decrease in the single-channel conductance of TRPV1

Abstract

TRPV1 is a Ca²⁺ permeable cation channel gated by multiple stimuli including noxious heat, capsaicin, protons, and extracellular cations. In this paper, we show that Ca²⁺ causes a concentration and voltage-dependent decrease in the capsaicin-gated TRPV1 single-channel conductance. This Ca²⁺-dependent effect on conductance was strongest at membrane potentials between −60 and +20 mV, but was diminished at more hyperpolarised potentials. Using simultaneous recordings of membrane current and fura-2 fluorescence to measure the fractional Ca²⁺ current of whole-cell currents evoked through wild-type and mutant TRPV1, we investigated a possible link between the mechanisms underlying Ca²⁺ permeation and the Ca²⁺-dependent effect on conductance. Surprisingly, we found no evidence of a structural correlation, and observed that the substitution of amino acids known to regulate Ca²⁺ permeability had little effect on the ability for Ca²⁺ to decrease TRPV1 conductance. However, we did observe that the Ca²⁺-dependent effect on conductance was not diminished by negative hyperpolarisation for a mutant receptor with severely impaired Ca²⁺ permeability, TRPV1-D646N/E648Q/E651Q. This would be consistent with the idea that Ca²⁺ reduces conductance by interacting with an intra-pore binding site, and that negative hyperpolarization reduces occupancy of this site by speeding the exit of Ca²⁺ into the cell. Taken together, our data show that in addition to directly and indirectly regulating channel gating, Ca²⁺ also directly reduces the conductance of TRPV1. Surprisingly, the mechanism underlying this Ca²⁺-dependent effect on conductance is largely independent of mechanisms governing Ca²⁺ permeability.