Determinants of Molecular Specificity in Phosphoinositide Regulation

Abstract

Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P 2 ) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P 3 ) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P 2 -specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P 3 -specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P 2 inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P 2 , and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.Although they make up only 1-5% of the total anionic lipids in a cell membrane (1-3), phosphoinositides have a prominent role in lipid signaling (4). Both phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P 2 ) 3 and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P 3 ) have been implicated in protein trafficking, cell motility, Ca 2ϩ signaling, cell growth, endo-and exocytosis, and ion transporter and channel regulation (4, 5). Changes in the plasma membrane PI(4,5)P 2 concentration that result from activation of cell surface receptors are believed to regulate the function of many ion channels and transporters, including K ϩ channels, voltage-gated Ca 2ϩ channels, inositol trisphosphate receptors, and the family of transient receptor potential (TRP) channels (4).Several different phosphoinositides have been implicated in modulation of TRP channels. The original TRP channel from Drosophila is thought to be gated by either diacyl glycerol or a polyunsaturated fatty acid (6). This is in contrast to original studies that implied an inhibitory role for PI(4,5)P 2 on both TRP and TRPL. A recent study demonstrated phosphoinositide binding by TRPC6, a TRP channel involved in vasodilation, but did not determine whether PI(4,5)P 2 or PI(3,4,5)P 3 was the endogenous ligand (7). Finally, it has been shown that PI(4,5)P 2 can alter the function of TRPM4, TRPM5, TRPM8, TRPM7, and TRPV5 (8 -14), but whether PI(4,5)P 2 is indeed the physiological regulator has not been addressed.For the transient receptor potential vanilloid-type 1 (TRPV1) channel responsible for transduction of painful thermal...

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

“…In excised patches from cells in which VSP was coexpressed with TRPV1-WT, the VSPactivating depolarizations reduced the capsaicin-activated current (Fig. 4, A [top right], B, and C), as previously reported (Klein et al, 2008). In excised patches from cells coexpressing VSP with TRPV1-R721A and subjected to the VSP-activating voltage protocol, the fraction of the initial current remaining at the end of the VSP-activating protocol (I/I initial ) was significantly different for TRPV1-R721A compared with TRPV1-WT (Fig.…”

“…PI(4,5)P 2 has been proposed to inhibit TRPV1, mediating the sensitizing actions of bradykinin and nerve growth factor (Chuang et al, 2001;Cao et al, 2013a). In contrast, others (including us) have proposed that PI(4,5)P 2 activates TRPV1 (Stein et al, 2006;Lukacs et al, 2007Lukacs et al, , 2013aKlein et al, 2008), and the depletion of PI(4,5)P 2 by phospholipase C may play a role in Ca 2+ -dependent desensitization (Mercado et al, 2010;Lukacs et al, 2013b). However, a recent study measured the apparent affinity of TRPV1 for diC8-PI(4,5)P 2 and found that the activating effects occurred with a K 1/2 of only 0.03 mol % in the plasma membrane, raising the question of whether PI(4,5)P 2 levels in the plasma membrane ever get sufficiently low selectivity must involve multiple, specific inter actions between TRPV1 and the phosphoinositide ligand.…”

“…We have used this system previously to demonstrate a temporal correlation between depletion of membrane PI(4,5)P 2 and inhibition of TRPV1-WT capsaicinactivated currents (Klein et al, 2008). In the current study we used the same approach to compare the effect that the conversion of PI(4,5)P 2 into PI(4)P has on TRPV1-WT and TRPV1-R721A capsaicin-activated currents in whole cells.…”

“…Simultaneous perforated patch whole-cell voltage clamp and confocal imaging Experiments were performed at room temperature as previously described (Klein et al, 2008). In brief, filamented borosilicate glass pipettes heat polished with a microforge (3.0-4.0 MΩ) were filled with intracellular saline solution containing (in mM) 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl 2 , 0.05 EGTA, and 10 HEPES, pH 7.2, and 1 mg/ml amphotericin-B.…”

The Ubiquitin Ligase MYCBP2 Regulates Transient Receptor Potential Vanilloid Receptor 1 (TRPV1) Internalization through Inhibition of p38 MAPK Signaling

“…It has also been reported that Ca 2ϩ /calmodulin (Ca 2ϩ /CaM), an ubiquitous calcium sensor, may play a role in TRPV1 Ca 2ϩ -dependent desensitization by binding to the N-terminal ankyrin repeat domain (49,50). In addition it has been shown that Ca 2ϩ influx through TRPV1 activates a Ca 2ϩ -sensitive phospholipase C causing depletion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) during desensitization, and that the recovery of the channel from desensitization requires resynthesis of PIP 2 (51)(52)(53)(54).…”