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PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.

ABSTRACT: Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLC? Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.

SUBMITTER: Itsuki K

PROVIDER: S-EPMC4001779 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.

Itsuki Kyohei K Imai Yuko Y Hase Hideharu H Okamura Yasushi Y Inoue Ryuji R Mori Masayuki X MX

The Journal of general physiology 20140201 2

Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonanc ...[more]

PMID: 24470487

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Project description:Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) regulates activities of numerous ion channels including inwardly rectifying potassium (K(ir)) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K(V)) channels might be regulated by PI(4,5)P(2). Wide expression of K(V) channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K(V) channels by PI(4,5)P(2), we have coexpressed several of them in tsA-201 cells with a G protein-coupled receptor (M(1)R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P(2) with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P(2) at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K(V)7.1, K(V)7.2/7.3, and K(ir)2.1 channel current by 90-95%. Activation of M(1)R inhibited K(V)7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P(2) regulation of activity of K(V)1.1/K(V)β1.1, K(V)1.3, K(V)1.4, and K(V)1.5/K(V)β1.3, K(V)2.1, K(V)3.4, K(V)4.2, K(V)4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K(V)1.1/K(V)β1.1 and K(V)3.4, resulting in up-regulation of current density upon activation of M(1)R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P(2) at the plasma membrane by enzymes does not seem to influence activity of most tested K(V) channels, whereas it does strongly inhibit members of the K(V)7 and K(ir) families.

Project description:Low-threshold voltage-gated M-type potassium channels (M channels) are tetraheteromers, commonly of two Kv7.2 and two Kv7.3 subunits. Though gated by voltage, the channels have an absolute requirement for binding of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) to open. We have investigated the quantitative relation between the concentration of a water-soluble PI(4,5)P(2) analog, dioctanoyl-PI(4,5)P(2) (DiC(8)-PI(4,5)P(2)), and channel open probability (P(open)) by fast application of increasing concentrations of DiC(8)-PI(4,5)P(2) to the inside face of membrane patches excised from Chinese hamster ovary cells expressing M channels as heteromeric Kv7.2/7.3 subunits. The rationale for the experiments is that this will mimic the effect of changes in membrane PI(4,5)P(2) concentration. Single-channel conductances from channel current-voltage relations in cell-attached mode were 9.2 ± 0.1 pS with a 2.5-mM pipette [K(+)]. Plots of P(open) against DiC(8)-PI(4,5)P(2) concentration were best fitted using a two-component concentration-P(open) relationship with high and low affinity, half-maximal effective concentration (EC(50)) values of 1.3 ± 0.14 and 75.5 ± 2.5 µM, respectively, and Hill slopes of 1.4 ± 0.06. In contrast, homomeric channels from cells expressing only Kv7.2 or Kv7.3 constructs yielded single-component curves with EC(50) values of 76.2 ± 19.9 or 3.6 ± 1.0 µM, respectively. When wild-type (WT) Kv7.2 was coexpressed with a mutated Kv7.3 subunit with >100-fold reduced sensitivity to PI(4,5)P(2), the high-affinity component of the activation curve was lost. Fitting the data for WT and mutant channels to an activation mechanism with independent PI(4,5)P(2) binding to two Kv7.2 and two Kv7.3 subunits suggests that the two components of the M-channel activation curve correspond to the interaction of PI(4,5)P(2) with the Kv7.3 and Kv7.2 subunits, respectively, that channels can open when only the two Kv7.3 subunits have bound DiC(8)-PI(4,5)P(2), and that maximum channel opening requires binding to all four subunits.

Dataset Information

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.

Publications

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.

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