Agonist-induced changes in Ca²⁺ permeation through the nociceptor cation channel TRPA1

The Ca²⁺-permeable cation channel TRPA1 acts as an ionotropic receptor for various pungent compounds and as a noxious cold sensor in sensory neurons. It is unclear what proportion of the TRPA1-mediated current is carried by Ca²⁺ ions and how the permeation pathway changes during stimulation. Here, based on the relative permeability of the nonstimulated channel to cations of different size, we estimated a pore diameter of ∼11 Å. Combined patch-clamp and Fura-2 fluorescence recordings revealed that with 2 mM extracellular Ca²⁺, and at a membrane potential of -80 mV, ∼17% of the inward TRPA1 current is carried by Ca²⁺. Stimulation with mustard oil evoked an apparent dilatation of the pore of 3 Å and an increase in divalent cation selectivity and fractional Ca²⁺ current. Mutations in the putative pore that reduced the divalent permeability and fractional Ca²⁺ current also prevented mustard-oil-induced increases in Ca²⁺ permeation. It is interesting that fractional Ca²⁺ currents for wild-type and mutant TRPA1 were consistently higher than values predicted based on biionic reversal potentials using the Goldman-Hodgkin-Katz equation, suggesting that binding of Ca²⁺ in the pore hinders monovalent cation permeation. We conclude that the pore of TRPA1 is dynamic and supports a surprisingly large Ca²⁺ influx.