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A Cholesterol Dimer Stabilizes the Inactivated State of an Inward-Rectifier Potassium Channel.

ABSTRACT: Cholesterol oligomers reside in multiple membrane protein X-ray crystal structures. Yet, there is no direct link between these oligomers and a biological function. Here we present the structural and functional details of a cholesterol dimer that stabilizes the inactivated state of an inward-rectifier potassium channel KirBac1.1. K⁺ efflux assays confirm that high cholesterol concentration reduces K⁺ conductance. We then determine the structure of the cholesterol-KirBac1.1 complex using Xplor-NIH simulated annealing calculations driven by solid-state NMR distance measurements. These calculations identified an α-α cholesterol dimer docked to a cleft formed by adjacent subunits of the homotetrameric protein. We compare these results to coarse grain molecular dynamics simulations. This is one of the first examples of a cholesterol oligomer performing a distinct biological function and structural characterization of a conserved promiscuous lipid binding region.

SUBMITTER: Borcik CG

PROVIDER: S-EPMC8957755 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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A Cholesterol Dimer Stabilizes the Inactivated State of an Inward-Rectifier Potassium Channel.

Borcik Collin G CG Eason Isaac R IR Yekefallah Maryam M Amani Reza R Han Ruixian R Vanderloop Boden H BH Wylie Benjamin J BJ

Angewandte Chemie (International ed. in English) 20220209 13

Cholesterol oligomers reside in multiple membrane protein X-ray crystal structures. Yet, there is no direct link between these oligomers and a biological function. Here we present the structural and functional details of a cholesterol dimer that stabilizes the inactivated state of an inward-rectifier potassium channel KirBac1.1. K<sup>+</sup> efflux assays confirm that high cholesterol concentration reduces K<sup>+</sup> conductance. We then determine the structure of the cholesterol-KirBac1.1 c ...[more]

PMID: 34985791

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Project description:Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K(+) currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K(+) channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.

Dataset Information

A Cholesterol Dimer Stabilizes the Inactivated State of an Inward-Rectifier Potassium Channel.

Publications

A Cholesterol Dimer Stabilizes the Inactivated State of an Inward-Rectifier Potassium Channel.

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