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Phosphorylation regulates the sensitivity of voltage-gated Kv7.2 channels towards phosphatidylinositol-4,5-bisphosphate.

ABSTRACT:

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Phosphatidylinositol-4,5-bisphosphate (PIP₂ ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP₂ -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP₂ . Dephosphorylation of Kv7.2 affected channel inhibition via M₁ muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP₂ , thereby ensuring the tight regulation of the channel via G protein-coupled receptors.

Abstract

The function of numerous ion channels is tightly controlled by G protein-coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol-4,5-bisphosphate (PIP₂ ). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP₂ and through phosphorylation. Using liquid chromatography-coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2-binding domains. To evaluate the effect of phosphorylation on PIP₂ -mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A⁵ mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP₂ depletion via the voltage-sensitive phosphatase Dr-VSP than were wild-type channels. In vitro phosphorylation assays with the purified C-terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild-type but not mutant Kv7.2 channels towards PIP₂ depletion via Dr-VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M₁ receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP₂ -binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR-mediated channel regulation.

SUBMITTER: Salzer I

PROVIDER: S-EPMC5215842 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Kv7 K(+)-channel subunits differ in their apparent affinity for PIP(2) and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP(2) affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M(1) receptors by oxo-M leads to PIP(2) depletion through G(q) and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M(1) receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC(50) for current suppression was 0.44 +/- 0.08 microM, and the maximal inhibition (Inhib(max)) was 74 +/- 3% (n = 5-7). When tonic PIP(2) abundance was increased by overexpression of PIP 5-kinase, the EC(50) was shifted threefold to the right (1.2 +/- 0.1 microM), but without a significant change in Inhib(max) (73 +/- 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3(T)) that increases whole-cell currents by 30-fold without any change in apparent PIP(2) affinity. Kv7.3(T) currents had a slightly right-shifted EC(50) as compared with Kv7.2/7.3 heteromers (1.0 +/- 0.8 microM) and a strongly reduced Inhib(max) (39 +/- 3%). In contrast, the dose-response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 +/- 8 nM), and Inhib(max) was slightly increased (81 +/- 6%, n = 3-4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP(2) by coexpressing them with Kv7.3(T) subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3(T)) or higher affinity (Kv7.2 (R463E)/Kv7.3(T)) channels, the EC(50) and Inhib(max) were similar to Kv7.4 or Kv7.3(T) homomers (0.12 +/- 0.08 microM and 79 +/- 6% [n = 3-4] and 0.58 +/- 0.07 microM and 27 +/- 3% [n = 3-4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3(T). The resulting heteromer displayed a very low EC(50) for inhibition (32 +/- 8 nM) and a slightly increased Inhib(max) (83 +/- 3%, n = 3-4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP(2) hydrolysis, PIP(2) binding to Kv7-channel subunits, and K(+) current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP(2) affinities.

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Phosphorylation regulates the sensitivity of voltage-gated Kv7.2 channels towards phosphatidylinositol-4,5-bisphosphate.

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