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Calmodulin confers calcium sensitivity to the stability of the distal intracellular assembly domain of Kv7.2 channels.

ABSTRACT: Tetrameric coiled-coil structures are present in many ion channels, often adjacent to a calmodulin (CaM) binding site, although the relationship between the two is not completely understood. Here we examine the dynamic properties of the ABCD domain located in the intracellular C-terminus of tetrameric, voltage-dependent, potassium selective Kv7.2 channels. This domain encompasses the CaM binding site formed by helices A and B, followed by helix C, which is linked to the helix D coiled-coil. The data reveals that helix D stabilizes CaM binding, promoting trans-binding (CaM embracing neighboring subunits), and they suggest that the ABCD domain can be exchanged between subunits of the tetramer. Exchange is faster when mutations in AB weaken the CaM interaction. The exchange of ABCD domains is slower in the presence of Ca²⁺, indicating that CaM stabilization of the tetrameric assembly is enhanced when loaded with this cation. Our observations are consistent with a model that involves a dynamic mechanism of helix D assembly, which supports reciprocal allosteric coupling between the A-B module and the coiled-coil formed by the helix D. Thus, formation of the distal helix D tetramer influences CaM binding and CaM-dependent Kv7.2 properties, whereas reciprocally, CaM and Ca²⁺ influence the dynamic behavior of the helix D coiled-coil.

SUBMITTER: Alaimo A

PROVIDER: S-EPMC5647379 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Calmodulin confers calcium sensitivity to the stability of the distal intracellular assembly domain of Kv7.2 channels.

Alaimo Alessandro A Nuñez Eider E Aivar Paloma P Fernández-Orth Juncal J Gomis-Perez Carolina C Bernardo-Seisdedos Ganeko G Malo Covadonga C Villarroel Alvaro A

Scientific reports 20171018 1

Tetrameric coiled-coil structures are present in many ion channels, often adjacent to a calmodulin (CaM) binding site, although the relationship between the two is not completely understood. Here we examine the dynamic properties of the ABCD domain located in the intracellular C-terminus of tetrameric, voltage-dependent, potassium selective Kv7.2 channels. This domain encompasses the CaM binding site formed by helices A and B, followed by helix C, which is linked to the helix D coiled-coil. The ...[more]

PMID: 29044210

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Project description:Key pointsPhosphatidylinositol-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.AbstractThe 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.

Dataset Information

Calmodulin confers calcium sensitivity to the stability of the distal intracellular assembly domain of Kv7.2 channels.

Publications

Calmodulin confers calcium sensitivity to the stability of the distal intracellular assembly domain of Kv7.2 channels.

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