Phosphoinositide interacting regulator of TRP (Pirt) enhances TRPM8 channel activity in vitro via increasing channel conductance

Abstract

Aim: Pirt is a two-transmembrane domain protein that regulates the function of a variety of ion channels. Our previous study indicated that Pirt acts as a positive endogenous regulator of the TRPM8 channel. The aim of this study was to investigate the mechanism underlying the regulation of TRPM8 channel by Pirt. Methods: HEK293 cells were transfected with TRPM8+Pirt or TRPM8 alone. Menthol (1 mmol/L) was applied through perfusion to induce TRPM8-mediated voltage-dependent currents, which were recorded using a whole-cell recording technique. PIP2 (10 μmol/L) was added into the electrode pipettes (PI was taken as a control). Additionally, cell-attached single-channel recordings were conducted in CHO cells transfected with TRPM8+Pirt or TRPM8 alone, and menthol (1 mmol/L) was added into the pipette solution. Results: Either co-transfection with Pirt or intracellular application of PIP2 (but not PI) significantly enhanced menthol-induced TRPM8 currents. Furthermore, Pirt and PIP2 synergistically modulated menthol-induced TRPM8 currents. Single-channel recordings revealed that co-transfection with Pirt significantly increased the single channel conductance. Conclusion: Pirt and PIP2 synergistically enhance TRPM8 channel activity, and Pirt regulates TRPM8 channel activity by increasing the single channel conductance.

Phosphoinositide-interacting regulator of TRP (PIRT) has opposing effects on human and mouse TRPM8 ion channels

“…3A). This agrees with previous similar studies of the mouse orthologs, which show that mouse TRPM8 membrane trafficking is also not affected by mouse PIRT coexpression and that mouse PIRT increases single channel conductance (12)(13)(14).…”

“…Cell-surface trafficking experiments indicate that enhanced channel activity in mice is not caused by changes in expression levels or channel trafficking, supporting the idea that PIRT enhances channel activity by interacting directly with the channels rather than increasing the number of channels in the membrane (12,14). More recently, it was shown that PIRT works synergistically with PIP 2 to enhance TRPM8 currents by increasing conductance at the single channel level (13). However, the details of this interaction including the binding site and stoichiometry of PIRT to TRPM8 are unknown.…”

“…TPRM8 activity is also modulated by a phosphoinositideinteracting regulator of TRP (PIRT), a small two-span membrane protein (12,13). PIRT was originally identified as a regulator of TRPV1, and it is exclusively expressed in dorsal root ganglia, trigeminal ganglia, and enteric neurons of the peripheral nervous system (14).…”

TRPM8 is required for survival and radioresistance of glioblastoma cells

“…TRPM8 channel function is reportedly modulated by G-protein coupled receptors [ 34 ], phosphoinosites [ 15 ], serine/threonine [ 35 ] and tyrosine kinases/phosphatases [ 36 ], as well as UBA1-dependent ubiquitination [ 19 ]. Moreover, protein-protein interactions with the two-transmembrane domain protein Pirt [ 37 ] and the TRP channel-associated factors (TCAFs) [ 38 ] reportedly promote trafficking and/or modulate activity of TRPM8. Finally, picomolar concentrations of testosterone have been shown to stimulate openings of the purified TRPM8 channel protein in planar lipid bilayers [ 16 ].…”

PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands

“…We show that PIRT specifically binds oxytocin. It is plausible that PIRT is involved with oxytocin regulation given that the oxytocin receptor is a G qα type G protein-coupled receptor (GPCR), G qα GPCRs directly influence PIP 2 levels, GPCRs are implicated with TRP channels, and PIRT is a PIP 2 binding protein that functionally modulates TRP channels [12][13][14]23,25,[76][77][78]. Similarly, PIRT was shown to be important for uterine contraction pain under oxytocin insult during birth in mice [14].…”