Unnatural amino acid photo-crosslinking of the IKs channel complex demonstrates a KCNE1:KCNQ1 stoichiometry of up to 4:4

ELife
Christopher I MurrayDavid Fedida

Abstract

Cardiac repolarization is determined in part by the slow delayed rectifier current (IKs), through the tetrameric voltage-gated ion channel, KCNQ1, and its β-subunit, KCNE1. The stoichiometry between α and β-subunits has been controversial with studies reporting either a strict 2 KCNE1:4 KCNQ1 or a variable ratio up to 4:4. We used IKs fusion proteins linking KCNE1 to one (EQ), two (EQQ) or four (EQQQQ) KCNQ1 subunits, to reproduce compulsory 4:4, 2:4 or 1:4 stoichiometries. Whole cell and single-channel recordings showed EQQ and EQQQQ to have increasingly hyperpolarized activation, reduced conductance, and shorter first latency of opening compared to EQ - all abolished by the addition of KCNE1. As well, using a UV-crosslinking unnatural amino acid in KCNE1, we found EQQQQ and EQQ crosslinking rates to be progressively slowed compared to KCNQ1, which demonstrates that no intrinsic mechanism limits the association of up to four β-subunits within the IKs complex.

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Citations

Mar 29, 2019·Proceedings of the National Academy of Sciences of the United States of America·Maartje WesthoffDavid Fedida
May 17, 2019·Essays in Biochemistry·Alexander R NödlingYu-Hsuan Tsai
Jul 11, 2019·Nature Reviews. Neuroscience·Pierre PaolettiAlexandre Mourot
Jul 28, 2020·The Journal of Physiology·N BraunS A Pless
Jul 9, 2017·The Journal of General Physiology·Emely ThompsonDavid Fedida
Nov 20, 2016·The Journal of Physiology·Eleonora GrandiVladimir Yarov-Yarovoy
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Jun 20, 2019·Proceedings of the National Academy of Sciences of the United States of America·Mette H PoulsenAndrew J R Plested
Dec 14, 2019·American Journal of Physiology. Heart and Circulatory Physiology·Jerri ChenThomas V McDonald
Jun 26, 2020·Frontiers in Physiology·Yundi WangDavid Fedida
Nov 10, 2020·ELife·Shawn M LamotheHarley T Kurata
Jul 16, 2020·Annual Review of Pharmacology and Toxicology·Emely ThompsonDavid Fedida
Dec 30, 2020·Cell·Pablo Ávalos PradoGuillaume Sandoz
Dec 31, 2019·Cell·Ji Sun, Roderick MacKinnon
Oct 13, 2021·The Journal of General Physiology·Jodene EldstromDavid Fedida

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Methods Mentioned

BETA
fluorescence microscopy
immunoprecipitation
co-immunoprecipitation
transgenic
transfections
transfection

Software Mentioned

GraphPad
pClamp10
Prism
Clampfit

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