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Regulation of TRPV1 ion channel by phosphoinositide (4,5)-bisphosphate: the role of membrane asymmetry.

ABSTRACT: Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P2 in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P2 was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P2 in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P2 in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P2 whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P2 must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P2 may resolve the long standing controversy about whether PI(4,5)P2 is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.

SUBMITTER: Senning EN

PROVIDER: S-EPMC4036241 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Publications

Regulation of TRPV1 ion channel by phosphoinositide (4,5)-bisphosphate: the role of membrane asymmetry.

Senning Eric N EN Collins Marcus D MD Stratiievska Anastasiia A Ufret-Vincenty Carmen A CA Gordon Sharona E SE

The Journal of biological chemistry 20140305 16

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 curren ...[more]

PMID: 24599956

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Project description:Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K+ channels and voltage-gated Ca2+ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K+ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K+ channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP2 strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP2 has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4-S5 linker) and R479 near the S6 activation gate are required for PIP2 to inhibit voltage activation. The ability of PIP2 to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP2 on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP2-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP2 can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP2 regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.

Dataset Information

Regulation of TRPV1 ion channel by phosphoinositide (4,5)-bisphosphate: the role of membrane asymmetry.

Publications

Regulation of TRPV1 ion channel by phosphoinositide (4,5)-bisphosphate: the role of membrane asymmetry.

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