Selectivity of ionic pathways through TRPM3 channel variants and mutants

Many different isoforms of TRPM3 proteins have been found. Channels made from the predominant isoform, TRPM3alpha2, can be activated by endogenous steroids such as pregnenolone sulfate and are highly permeable to divalent cations. An alternatively spliced isoform with an altered pore region, TRPM3alpha1, produces channels with distinctly different pharmacological and biophysical properties: They have a higher constitutive activity, a differently shaped current-voltage relationship and lower relative permeabilities for divalent cations. Pharmacologically, TRPM3alpha1 channels can be activated by the antifungal compound clotrimazole, but not by pregnenolone sulfate. Interestingly, clotrimazole has previously been reported to activate a separate ionic conduction pathway through the voltage-sensor-like domain of TRPM3alpha2, but not of TRPM3alpha1 channels. This non-canonical, omega pore has also been reported to be active in a disease-causing mutant of TRPM3alpha2 and to strongly prefer Na+ over divalent cations. Using different TRPM3 isoforms and mutants thereof overexpressed in HEK293 cells, we directly compared the ionic selectivity of the different pores of TRPM3 channels by using appropriate pharmacological agents to measure reversal potentials under a variety of bi-ionic conditions. Consistent with earlier findings, our results show that TRPM3alpha1 channels have a much lower relative permeability for divalent cations compared to TRPM3alpha2 channels. The selectivity of the channels did not depend on the nature of the agonists applied under all conditions tested, neither in TRPM3alpha1 nor in TRPM3alpha2 channels. However, the biophysical properties (including the ionic selectivity) of TRPM3 channels are dramatically altered by mutating single residues in the central pore. Thus, our results favor a model in which the selectivity filter of TRPM3 channels is only determined by residues in the central pore of the tetrameric channel structures. Consequently, our data do not support the existence of non-canonical omega-pores activated by clotrimazole in TRPM3 channels.