The human potassium channel KCNQ1 is a polytopic ?-helical membrane protein. KCNQ1 is expressed in both epithelial and heart tissue. In the heart, KCNQ1, in association with KCNE1, mediates the Iks current responsible for the repolarization of the cardiac action potential. Mutations that cause a loss-of-function of KCNQ1 result in a congenital condition known as long-QT syndrome (LQTS). Congenital LQTS predisposes an individual to cardiac arrhythmia and can result in sudden death. This proposal focuses on a single mutation, S225L, which is sufficient to cause LQTS by significantly altering the voltage-response of KCNQ1. The conductance-voltage (G-V) relationship of S225L is shifted relative to wild type such that much higher voltages are required for channel conductance. Homology models suggest that S225 undergoes a significant change in environment, moving from a hydrophobic to a more polar environment, as KCNQ1 transitions from the closed- to open-state. While current homology models are sufficient to generate a testable hypothesis, low sequence conservation between KCNQ1 and other potassium channel structures (~20%) highlight the need for an experimentally determined KCNQ1 structure. The central hypothesis of this proposal is that the closed- state of the KCNQ1 voltage-senor is stabilized by the hydrophobic character of the S225L substitution. This hypothesis will be tested in two ways. First, by introducing a series of amino acid substitutions at position 225 that vary i charge, size, and hydrophobicity. It is expected that closed-state stabilization will correlate wit increasing hydrophobic character of the amino acid substitution. The effects of these mutations will be evaluated in both the full KCNQ1 channel, and in the isolated voltage-senor domain (VSD). Using nuclear magnetic resonance (NMR) spectroscopy the open-closed equilibrium of VSD in the absence of an electric field can be measured. Published state-locking mutations will be used to provide homogeneous NMR reference spectra for each state, thus allowing the open-closed equilibrium of a given VSD mutation to be directly measured. Using planar-patch-camp methods the G-V curve of the KCNQ1 channel can be measured. This approach will allow for the effects of the mutational series to be evaluated in the context of the complete channel. It is expected that changes observed for the isolated VSD will be correlated with that of the full channel. Second, the closed-state structure of the VSD, using state-locking mutations, will be determined by NMR. The combination of electrophysiology, biophysical characterization, and closed-state structure will establish the biochemical underpinnings of the LQTS-inducing S225L mutation. The closed-state structure of the VSD would represent a significant contribution to the field providing insight into the mechanism of the open- closed transition.
The human potassium channel KCNQ1 mediates the Iks current in cardiac myocytes, playing a central role in repolarization of the cardiac action potential. A single mutation in KCNQ1, S225L, is sufficient to cause a loss- of-function that results in a congenital condition known as long-QT syndrome, which predisposes an individual for cardiac arrhythmias. Our goal is determine the structure of the voltage sensor domain of KCNQ1 in its closed state and develop a model by which S225L causes loss-of-function and hence long-QT syndrome.
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