Potassium (K+) channels are major determinants of cell excitability and play crucial roles in many physiological processes. Central to a mechanistic understanding of ion channels is the process of gating, the opening and closing of the pore to ionic flow. Gating malfunctions can have disastrous physiological consequences. The overall objective of this grant is to understand the molecular mechanisms of ligand modulation and gating in K+ channels by employing functional, structural, and theoretical analysis on model prokaryotic K+ channels, homologues of eukaryotic K+ channels. Bacterial channels lend themselves to the biochemical and structural studies necessary to investigate these mechanisms. Our first major aim is to understand ligand gating in K+ channels using KcsA as a model.
In Aims 1. 1 and 1.2 we propose to determine directly the pKas (the intrinsic proton binding affinities) of two proposed proton-sensing residues using NMR spectroscopy, in both open and closed states. We will take advantage of mutants with changed pH gating as determined with electrophysiology to guide interpretations of peak assignments and chemical shift changes in the NMR spectra. We will also use crosslinked closed and open KcsA constructs that will allow us to determine pKas of pH sensing residues in the absence of the conformational change that gates the channel. To our knowledge, this would be the first direct determination of intrinsic ligand binding affinities to both open and closed states.
In Aim 1. 3 we will formulate a Monod-Wyman-Changeux model for proton gating in KcsA constrained with results from the combined approach of modeling, mutagenesis, electrophysiology, X-ray crystallography and NMR. X-ray crystallography will be used to structurally determine the effect of channel mutations. Our second major aim is to understand gating of Ca2+-activated K+ channels using MthK as a model.
Aims 2. 1 and 2.2 investigate the physical location of the gate modulated by Ca2+. We will investigate whether the MthK Ca2+ activation gate is at the bundle crossing or at the selectivity filter by determining if the access of intracellular blockers that bnd inside the vestibule of K+ channels (between the two gates) depends on whether the channel is open or closed. Accessibility of the blockers to the vestibule in the absence of Ca2+ is an indication of a selectivity filter gate.
In Aim 2. 3 we propose to investigate MthK slow desensitization, a phenomenon observed only in liposomes, not in bilayer recordings, by altering the bilayer composition. This phenomenon is intriguing as its differential occurrence would indicate that a fundamental, physiologically relevant, channel property is digitally modulated by bilayer parameters. We will use a stopped-flow bulk assay that measures the quenching of a fluorophore upon Tl+ entry through MthK-reconstituted vesicles to determine the affinity and the kinetics of blockers both in the presence and absence of Ca2+ as well as determine the effect of lipids on desensitization. The proposed aims will provide new insights into the fundamental K+ channel properties, which will be readily applicable to our understanding of the broader family of eukaryotic K+ channels.

Public Health Relevance

Ion channels are proteins that allow the conduction of ions across cell membranes, forming the basis of electrical signaling. Their proper function is of great importance as mutations in ion channel genes underlie numerous diseases and these channels are targets for a large number of drugs which help control conditions such as pain, hypertension, sleep disorders, psychiatric disorders. The proposed studies, applicable to an array of critical ion channel proteins, investigate the molecular mechanism behind how potassium channels open and close their pore, a process called gating, and how ligand binding affects gating.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088352-08
Application #
9382893
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Nie, Zhongzhen
Project Start
2010-09-10
Project End
2019-11-30
Budget Start
2017-12-01
Budget End
2019-11-30
Support Year
8
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Schmidpeter, Philipp A M; Gao, Xiaolong; Uphadyay, Vikrant et al. (2018) Ligand binding and activation properties of the purified bacterial cyclic nucleotide-gated channel SthK. J Gen Physiol 150:821-834
Schmidpeter, Philipp A M; Nimigean, Crina M (2018) Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels. Bio Protoc 8:
Rheinberger, Jan; Gao, Xiaolong; Schmidpeter, Philipp Am et al. (2018) Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures. Elife 7:
Posson, David J; Rusinova, Radda; Andersen, Olaf S et al. (2018) Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies. Methods Mol Biol 1684:223-235
Menny, Anaïs; Lefebvre, Solène N; Schmidpeter, Philipp Am et al. (2017) Identification of a pre-active conformation of a pentameric channel receptor. Elife 6:
Heer, Florian T; Posson, David J; Wojtas-Niziurski, Wojciech et al. (2017) Mechanism of activation at the selectivity filter of the KcsA K+ channel. Elife 6:
Kim, Dorothy M; Dikiy, Igor; Upadhyay, Vikrant et al. (2016) Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles. J Gen Physiol 148:119-32
Kim, Dorothy M; Nimigean, Crina M (2016) Voltage-Gated Potassium Channels: A Structural Examination of Selectivity and Gating. Cold Spring Harb Perspect Biol 8:
Posson, David J; Rusinova, Radda; Andersen, Olaf S et al. (2015) Calcium ions open a selectivity filter gate during activation of the MthK potassium channel. Nat Commun 6:8342
Rusinova, Radda; Kim, Dorothy M; Nimigean, Crina M et al. (2014) Regulation of ion channel function by the host lipid bilayer examined by a stopped-flow spectrofluorometric assay. Biophys J 106:1070-8

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