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J Biol Chem. 2018 Jun 15;293(24):9423-9434. doi: 10.1074/jbc.RA118.003563. Epub 2018 May 3.

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

Hilton JK1,2,3, Salehpour T1,2,3, Sisco NJ1,2,3, Rath P1,2,3, Van Horn WD4,2,3.

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From the School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287.
the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona 85281, and.
The Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287.
From the School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287,


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.


channel modulation; electrophysiology; ion channel; membrane protein; nuclear magnetic resonance (NMR); phosphoinositide; phosphoinositide regulator of TRP; transient receptor potential channels (TRP channels)

[Available on 2019-06-15]

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