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

Jacob K Hilton,Taraneh Salehpour,Nicholas J Sisco,Parthasarathi Rath,Wade D Van Horn

doi:10.1074/jbc.ra118.003563

Abstract

Transient receptor potential melastatin 8 (TRPM8) is a cold-sensitive ion channel with diverse physiological roles. TRPM8 activity is modulated by many mechanisms, including an interaction with the small membrane protein phosphoinositide-interacting regulator of TRP (PIRT). Here, using comparative electrophysiology experiments, we identified species-dependent differences between the human and mouse TRPM8-PIRT complexes. We found that human PIRT attenuated human TPRM8 conductance, unlike mouse PIRT, which enhanced mouse TRPM8 conductance. Quantitative Western blot analysis demonstrates that this effect does not arise from decreased trafficking of TRPM8 to the plasma membrane. Chimeric human/mouse TRPM8 channels were generated to probe the molecular basis of the PIRT modulation, and the effect was recapitulated in a pore domain chimera, demonstrating the importance of this region for PIRT-mediated regulation of TRPM8. Moreover, recombinantly expressed and purified human TRPM8 S1-S4 domain (comprising transmembrane helices S1-S4, also known as the sensing domain, ligand-sensing domain, or voltage sensing-like domain) and full-length human PIRT were used to investigate binding between the proteins. NMR experiments, supported by a pulldown assay, indicated that PIRT binds directly and specifically to the TRPM8 S1-S4 domain. Binding became saturated as the S1-S4:PIRT mole ratio approached 1. Our results have uncovered species-specific TRPM8 modulation by PIRT. They provide evidence for a direct interaction between PIRT and the TRPM8 S1-S4 domain with a 1:1 binding stoichiometry, suggesting that a functional tetrameric TRPM8 channel has four PIRT-binding sites.

Highlights

Coimmunoprecipitation experiments show that phosphoinositideinteracting regulator of TRP (PIRT) interacts with TRPV1 and Transient receptor potential melastatin 8 (TRPM8), suggesting that the regulation is mediated via a direct interaction with the channel [14]
At ambient room temperature (ϳ23 °C), recordings of cells transfected with both human TRPM8 and PIRT resulted in significantly reduced currents compared with cells transfected with human TRPM8 and an empty pIRES2–DsRed vector at voltages of ϩ60 mV and above; at ϩ100 mV, cells transfected with human PIRT showed on average ϳ69% of the current measured from cells expressing hTRPM8 alone
PIRT has previously been shown in behavioral knock-out mice studies to be important in thermosensation via regulation of the thermosensitive TRP channels TRPV1 and TRPM8, demonstrating that the biophysical effects of PIRT on TRPM8 are functionally significant at the organismal level [12, 14]

Summary

To whom correspondence should be addressed

TPRM8 activity is modulated by a phosphoinositideinteracting regulator of TRP (PIRT), a small two-span membrane protein [12, 13]. Transmembrane domain chimeras of human/mouse TRPM8 show that residues in the transmembrane domain (helices S1–S6), and in particular the pore domain (S5–S6), are the basis for modulation by PIRT and the species differences observed. These results shed light on the PIRT–TRPM8 interaction and reveal that the PIRT–TRPM8 complex has evolved in a species-dependent manner

Results

Discussion

Experimental procedures