Dataset Information

The I_Ks Channel Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry.

ABSTRACT: The delayed potassium rectifier current, I_Ks, 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 I_Ks 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 I_Ks 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 V_1/2 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 V_1/2 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.

SUBMITTER: Thompson E

PROVIDER: S-EPMC6224683 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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The IKs Channel Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry.

Thompson Emely E Eldstrom Jodene J Westhoff Maartje M McAfee Donald D Fedida David D

Biophysical journal 20180927 9

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 m ...[more]

PMID: 30314657

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Project description:The IKs channel complex is formed by the co-assembly of Kv7.1 (KCNQ1), a voltage-gated potassium channel, with its β-subunit, KCNE1 and the association of numerous accessory regulatory molecules such as PIP2, calmodulin, and yotiao. As a result, the IKs potassium current shows kinetic and regulatory flexibility, which not only allows IKs to fulfill physiological roles as disparate as cardiac repolarization and the maintenance of endolymph K+ homeostasis, but also to cause significant disease when it malfunctions. Here, we review new areas of understanding in the assembly, kinetics of activation and inactivation, voltage-sensor pore coupling, unitary events and regulation of this important ion channel complex, all of which have been given further impetus by the recent solution of cryo-EM structural representations of KCNQ1 alone and KCNQ1+KCNE3. Recently, the stoichiometric ratio of KCNE1 to KCNQ1 subunits has been confirmed to be variable up to a ratio of 4:4, rather than fixed at 2:4, and we will review the results and new methodologies that support this conclusion. Significant advances have been made in understanding differences between KCNQ1 and IKs gating using voltage clamp fluorimetry and mutational analysis to illuminate voltage sensor activation and inactivation, and the relationship between voltage sensor translation and pore domain opening. We now understand that the KCNQ1 pore can open with different permeabilities and conductance when the voltage sensor is in partially or fully activated positions, and the ability to make robust single channel recordings from IKs channels has also revealed the complicated pore subconductance architecture during these opening steps, during inactivation, and regulation by 1-4 associated KCNE1 subunits. Experiments placing mutations into individual voltage sensors to drastically change voltage dependence or prevent their movement altogether have demonstrated that the activation of KCNQ1 alone and IKs can best be explained using allosteric models of channel gating. Finally, we discuss how the intrinsic gating properties of KCNQ1 and IKs are highly modulated through the impact of intracellular signaling molecules and co-factors such as PIP2, protein kinase A, calmodulin and ATP, all of which modulate IKs current kinetics and contribute to diverse IKs channel complex function.

Project description:Key pointsIn adult ventricular myocytes, the slow delayed rectifier (IKs ) channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer.AbstractSlow delayed rectifier (IKs ) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β-adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to β-adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected IKs channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer.

Dataset Information

The I_Ks Channel Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry.

Publications

The I<sub>Ks</sub> Channel Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry.

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