Mutations in the voltage‐sensing domain affect the alternative ion permeation pathway in the TRPM3 channel

KEY POINTS: Mutagenesis at positively charged amino acids (arginines and lysines) (R1–R4) in the voltage‐sensor domain (transmembrane segment (S) 4) of voltage‐gated Na(+), K(+) and Ca(2+) channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non‐canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non‐canonical pore exists in the native TRPM3 channel and can be activated by co‐stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K(+) channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage‐sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non‐canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage‐sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. ABSTRACT: Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1–S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K(+) channels, in which mutations in S4 can create an analogous ‘omega’ pore, we performed site‐directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non‐canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway.