Exploring the gating mechanisms of the TRPM3 channel.

Abstract

TRP channels are polymodal sensors that detect a variety of physiological stimuli. TRPM3, a member of the TRP channel family, is a nonselective cation channel implicated in disorders throughout the body, including in diabetes, neurodevelopmental disorders, chronic inflammation, and chronic fatigue syndrome. As such, understanding TRPM3 function and dysfunction could provide valuable insight into physiological functioning and treatment. While TRPM3 has many isoforms, the isoform studied here is the most well-characterized TRPM3 isoform, TRPM3α2. TRPM3α2 is sensitive to heat, depolarizing membrane voltages, and ligands such as pregnenolone sulfate (PS). While PS is not specific for TRPM3, it is currently the most used agonist for the channel as it activates the channel at physiological concentrations and elicits an intracellular signaling cascade typical of TRPM3 stimulation. TRPM3α2 is also interesting from a mechanistic perspective because it has been proposed to have two ion permeation pathways, one at the central axis within the pore domain formed by S5-S6 helices and activated by PS, and a second activated by other agonists and located within S1-S4 domains that resemble voltage-sensing domains in voltage-activated potassium (Kv) channels. We began by identifying ionic conditions where we could measure macroscopic ionic currents during test depolarizations and tail currents upon membrane repolarization. We find that symmetrical CsCl based solutions seem to stabilize the open state of TRPM3, whether examining voltage-dependent activation at room temperature in the absence of other activators, or in the presence of PS, enabling robust resolution of tail current. Using tail current measurements, we observed that the conductance (G) - voltage (V) relationship for TRPM3 is complex, with distinct components at negative and positive membrane voltages. We are currently exploring ligands thought to activate the alternate permeation pathway and using mutagenesis to localize the PS binding site.