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Modulation of the IKS channel by PIP2 requires two binding sites per monomer

ABSTRACT: Highlights • Two PIP2 lipids are required to stabilize each Voltage Sensor Domain in the IKS channel.• The ancillary KCNE1 β-subunits to generate the IKS current interacts with both PIP2 binding sites.• The key KCNE1 basic residue involved in theses interactions are essential for modulation of the IKS PIP2 sensitivity.• The key KCNE1 basic residue involved in theses interactions are highly conserved across all members of the KCNE family. The phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipid has been shown to be crucial for the coupling between the voltage sensor and the pore of the potassium voltage-gated KV7 channel family, especially the KV7.1 channel. Expressed in the myocardium membrane, KV7.1 forms a complex with KCNE1 auxiliary subunits to generate the IKS current. Here we present molecular models of the transmembrane region of this complex in its three known states, namely the Resting/Closed (RC), the Intermediate/Closed (IC), and the Activated/Open (AO), robustness of which is assessed by agreement with a range of biophysical data. Molecular Dynamics (MD) simulations of these models embedded in a lipid bilayer including phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids show that in presence of KCNE1, two PIP2 lipids are necessary to stabilize each state. The simulations also show that KCNE1 interacts with both PIP2 binding sites, forming a tourniquet around the pore and preventing its opening. The present investigation provides therefore key molecular elements that govern the role of PIP2 in KCNE1 modulation of IKS channels, possibly a common mechanism by which auxiliary KCNE subunits might modulate a variety of other ion channels. Graphical abstract Image, graphical abstract

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PROVIDER: S-EPMC10074941 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Project description:The delayed potassium rectifier current, IKs, is assembled from tetramers of KCNQ1 and varying numbers of KCNE1 accessory subunits in addition to calmodulin. This channel complex is important in the response of the cardiac action potential to sympathetic stimulation, during which IKs is enhanced. This is likely due to channels opening more quickly, more often, and to greater sublevel amplitudes during adrenergic stimulation. KCNQ1 alone is unresponsive to cyclic adenosine monophosphate (cAMP), and thus KCNE1 is required for a functional effect of protein kinase A phosphorylation. Here, we investigate the effect that KCNE1 has on the response to 8-4-chlorophenylthio (CPT)-cAMP, a membrane-permeable cAMP analog, by varying the number of KCNE1 subunits present using fusion constructs of IKs with either one (EQQQQ) or two (EQQ) KCNE1 subunits in the channel complex with KCNQ1. These experiments use both whole-cell and single-channel recording techniques. EQQ (2:4, E1:Q1) shows a significant shift in V1/2 of activation from 10.4 mV ± 2.2 in control to -2.7 mV ± 1.2 (p-value: 0.0024). EQQQQ (1:4, E1:Q1) shows a smaller change in response to 8-CPT-cAMP, 6.3 mV ± 2.3 to -3.2 mV ± 3.0 (p-value: 0.0435). As the number of KCNE1 subunits is reduced, the shift in the V1/2 of activation becomes smaller. At the single-channel level, a similar graded change in subconductance occupancy and channel activity is seen in response to 8-CPT-cAMP: the less E1, the smaller the response. However, both constructs show a significant reduction of a similar magnitude in the first latency to opening (EQQ control: 0.90 s ± 0.07 to 0.71 s ± 0.06, p-value: 0.0032 and EQQQQ control: 0.94 s ± 0.09 to 0.56 s ± 0.07, p-value < 0.0001). This suggests that there are both E1-dependent and E1-independent effects of 8-CPT-cAMP on the channel.

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