bi0c00163_si_002.xlsx (81.21 kB)
Insight into the Selectivity of Kir3.2 toward Phosphatidylinositides
dataset
posted on 2020-05-21, 15:54 authored by Pei Qiao, Yang Liu, Tianqi Zhang, Amanda Benavides, Arthur LaganowskyActivation
of G-protein-gated inwardly rectifying potassium channels
(Kir3.x) requires the direct binding of phosphorylated
phosphatidylinositides (PIPs). Previous studies have established that
PIP isoforms activate Kir channels to varying degrees and the binding
affinity between PIPs and Kir3.2 appears to be correlated with the
level of activation. However, how individual residues contribute to
the selectivity of Kir channels toward PIP isoforms is poorly understood.
Here, we employ native mass spectrometry (MS) and fluorescent lipid
binding assays to gain insight into the contribution of specific Kir3.2
residues binding to phospholipids. For the wild-type channel, we demonstrate
the importance of membrane protein samples devoid of co-purified contaminants
for protein–lipid binding studies and show that PIP(4,5)P2 cooperatively binds Kir3.2 with a Hill coefficient of 2.7.
We also find lipid binding profiles determined from native MS and
solution binding assays are in direct agreement. Point mutations of
Kir3.2 residues that interact with PIPs distinctly alter selective
lipid binding. The K64Q mutation results in altered binding profiles
with the highest binding affinity for PIP(4,5)P2 with specific
acyl chains. Mutation of R92 to Pro, a residue found in Kir6.2, results
in promiscuous binding of PIP isoforms. Kir3.2 with the K194A mutation
results in a distinct binding preference for PIP(3,4,5)P3 over other PIP isoforms. Taken together, our results underscore
the utmost importance of protein quality for protein–lipid
binding studies and show that a single mutation in Kir3.2 can alter
the selectivity toward PIPs.