Inactivation occurs spontaneously after opening in all studied K+ channels, including model channels and the hERG channel that determines timing of the human heart. Inactivation controls channel signaling by determining mean open times and the delay before they can be re-opened, yet its molecular basis remains controversial, with several models proposed. We propose to clarify a key aspect distinguishing these models: is K+ ion release from the selectivity filter an essential step in inactivation? Also, it is hypothesizedto occur spontaneously because of transmembrane allosteric coupling: intracellular H+-triggered changes in the inner transmembrane helix, TM2, produce the conductive Activated state, but this creates clashes that destabilize the extracellular K+ loaded selectivity filter, and cause it o slowly decay to the K+ depleted state. KcsA, a proton-activated channel, provides a unique opportunity to understand this inactivation process in detail. In contrast to the X-ray diffractionor solution NMR studies, the proposed Solid State NMR studies will be performed on full- length KcsA in hydrated membrane bilayers, using wild type or mutants and varied buffer conditions;hence functional species identified in electrophysiology can be conveniently prepared for NMR. Key signatures for inactivation (mutation dependences, kinetics, and [K+] dependence) confirm that the low pH NMR-detected species is the Inactivated state. Mutants altered in inactivation will be used to further test whether K+ release is essential to inactivation. For several no inactivation is observed, and the dominant species at pH 3-5 is the Activated state. For others inactivation occurs quantitatively, and the dominant species at pH 3-5 is the Inactivated state. Comparing various mutants, correlation between inactivation (by electrophysiology) and K+ depletion (by NMR) will provide a clear test of our hypothesis. An initial study of wild type and E71A provides strong support for this hypothesis. Interconversion rates of the Resting, Activated and Inactivated from electrophysiology will be compared with the K+ release rates from NMR. Our recent 4D NMR data allow full spectral assignments, and show for the first time that the allosteric coupling operates in both directions: not only does protonation of the pH sensor cause K+ ion release at high ambient [K+], but also K+ ion extraction at low [K+] causes pH sensor protonation and opening of TM2 at neutral pH, which represents a novel mechanism for opening a K+ channel. NMR titrations will allow quantitative description of the allosteric coupling, clarifying of the role of the bilayer, and of key amino acids, using recently described coupling-impaired mutants, where bulky sidechains between the selectivity filter and the hinge of TM2 are removed. The high-resolution structure for the inactivated state has been elusive to date. High-quality NMR spectra of the inactivated state provide an excellent opportunity for structure determination in intact bilayers, as proposed herein.

Public Health Relevance

K+ channels serve as life's metronomes, dictating the timing for cardiac and nervous system events. The medical relevance of their inactivation processes is underscored by the fact that recently many highly promising drugs were abandoned because of adventitious binding to the hERG K+ channel in the heart, altering inactivation properties and causing acquired long QT syndrome and arrhythmia. We propose NMR experiments that will clarify the structure, stability, and dynamics of the inactivated state.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Chin, Jean
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Columbia University (N.Y.)
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New York
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Laage, Ségolène; Tao, Yisong; McDermott, Ann E (2015) Cardiolipin interaction with subunit c of ATP synthase: solid-state NMR characterization. Biochim Biophys Acta 1848:260-5
Wylie, Benjamin J; Dzikovski, Boris G; Pawsey, Shane et al. (2015) Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces. J Biomol NMR 61:361-7
Wylie, Benjamin J; Bhate, Manasi P; McDermott, Ann E (2014) Transmembrane allosteric coupling of the gates in a potassium channel. Proc Natl Acad Sci U S A 111:185-90
Bhate, Manasi P; Wylie, Benjamin J; Thompson, Ameer et al. (2013) Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay. Protein Expr Purif 91:119-24
Quinn, Caitlin M; McDermott, Ann E (2012) Quantifying conformational dynamics using solid-state Rýýýýý experiments. J Magn Reson 222:1-7
Bhate, Manasi P; McDermott, Ann E (2012) Protonation state of E71 in KcsA and its role for channel collapse and inactivation. Proc Natl Acad Sci U S A 109:15265-70
Siemer, Ansgar B; Huang, Kuo-Ying; McDermott, Ann E (2012) Protein linewidth and solvent dynamics in frozen solution NMR. PLoS One 7:e47242
Li, Wenbo; McDermott, Ann (2012) Investigation of slow molecular dynamics using R-CODEX. J Magn Reson 222:74-80
Huang, Kuo-Ying; Siemer, Ansgar B; McDermott, Ann E (2011) Homonuclear mixing sequences for perdeuterated proteins. J Magn Reson 208:122-7
Bauer, Andras J; Gieschler, Simone; Lemberg, Kathryn M et al. (2011) Functional model of metabolite gating by human voltage-dependent anion channel 2. Biochemistry 50:3408-10

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