Characterization of Transmembrane Potassium Channels and Their Lipid Environment via Solid-State Nuclear Magnetic Resonance Spectroscopy and Fluorescence Assays



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Potassium channels are regulated in part by allosteric interactions with the surrounding lipids of the membrane. The allosteric interactions correlate to long-range structural rearrangements necessary for channel function at both the selectivity filter and helical bundle crossing. Site-directed mutagenesis to investigate specific residues involved in the KirBac1.1 gating mechanism and the proposed allosteric network. We propose that these mutated residues are essential for the allosteric network, lipid-binding pocket, selectivity filter hydrogen bonding network, or protein-acyl chain interactions. We then use 13Clabeled lipids in the presence of the protein to determine the affinity between the anionic phospholipid PG and KirBac1.1 via SSNMR and additional functional assays. G protein-coupled inwardly rectifying potassium channels (GIRKs) are a natural evolution of these experiments. Activation of GIRK channels leads to a hyperpolarization of the neuron’s membrane potential, providing a fundamental component of inhibition required for nerve cell communication in both normal and diseased states. This includes the determination of the different states of the channel in multiple lipid environments. In this regime, after confirming the existence of multiple activated states using functional assays, we applied SSNMR spectroscopy to identify unique spectral signatures of multiple channel states.

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K+ channels,, SSNMR,, Fluorescence Assay,, FRET,, Allostery