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The Lipid Activation Mechanism of a Transmembrane Potassium Channel
journal contribution
posted on 2020-08-07, 15:10 authored by Collin
G. Borcik, Derek B. Versteeg, Reza Amani, Maryam Yekefallah, Nazmul H. Khan, Benjamin J. WylieMembrane proteins and lipids coevolved
to yield unique coregulatory
mechanisms. Inward-rectifier K+ (Kir) channels are often
activated by anionic lipids endemic to their native membranes and
require accessible water along their K+ conductance pathway.
To better understand Kir channel activation, we target multiple mutants
of the Kir channel KirBac1.1 via solid-state nuclear magnetic resonance
(SSNMR) spectroscopy, potassium efflux assays, and Förster
resonance energy transfer (FRET) measurements. In the I131C stability
mutant (SM), we observe an open-active channel in the presence of
anionic lipids with greater activity upon addition of cardiolipin
(CL). The introduction of three R to Q mutations (R49/151/153Q (triple
Q mutant, TQ)) renders the protein inactive within the same activating
lipid environment. Our SSNMR experiments reveal a stark reduction
of lipid–protein interactions in the TQ mutant explaining the
dramatic loss of channel activity. Water-edited SSNMR experiments
further determined the TQ mutant possesses greater overall solvent
exposure in comparison to wild-type but with reduced water accessibility
along the ion conduction pathway, consistent with the closed state
of the channel. These experiments also suggest water is proximal to
the selectivity filter of KirBac1.1 in the open-activated state but
that it may not directly enter the selectivity filter. Our findings
suggest lipid binding initiates a concerted rotation of the cytoplasmic
domain subunits, which is stabilized by multiple intersubunit salt
bridges. This action buries ionic side chains away from the bulk water,
while allowing water greater access to the K+ conduction
pathway. This work highlights universal membrane protein motifs, including
lipid–protein interactions, domain rearrangement, and water-mediated
diffusion mechanisms.
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Keywords
Kir channel KirBac 1.1selectivity filter131Cpotassium efflux assaysWater-edited SSNMR experimentswater-mediated diffusion mechanismsKir channel activationTransmembrane Potassium Channel Mem...lipidion conduction pathwaymembrane protein motifscytoplasmic domain subunitsCLFRETSMLipid Activation MechanismTQintersubunit salt bridges
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