The ultimate goal of the research described in this proposal is to fully characterize the properties of small conductance, apamin- blocked Ca-activated K channels (SK channels) that are probably responsible for the sustained afterhyperpolarization (AHP) in tissue cultured rat skeletal muscle. The kinetics, permeability properties and block of SK channels will be examined and the roles of SK channels and other Ca-activated K channels in muscle excitation will be determined. The modulation of SK channels by innervation will be studied, particularly addressing whether or not functional synapse formation is required for channel regulation. Currents through single SK channels will be recorded using the patch voltage clamp technique. Distributions of open and shut channel events will be fitted with sums of exponentials. The number of exponential components required to fit these distributions suggests a minimum number of the kinetic states that underlie SK channel activity. The apparent number of Ca ions that bind during activity will be determined by measuring the slopes of Hill plots of the percent of time the channel is open at several different intracellular Ca concentrations. Interval distributions predicted by several kinetic models will be compared with the experimentally observed distributions in order to find the models that are the most consistent with the experimental data. Permeability properties will be examined by several methods which will address the selectivity of SK channels as well as examine possible ion-channel interactions such as multiple ion occupancy. The block by apamin, a toxin that has a very high affinity for SK channels, will be characterized at the single channel level to investigate the mechanism of block. The roles of SK channels and large conductance Ca-activated channels (BK channels) will be examined by intracellular potential recording while, at the same time, monitoring single channel currents through a cell-attached membrane patch. With this technique it will be possible to observe which channels are active during the various phases of the action potential, particularly during the AHP. Neural modulation of SK channels will be studied by monitoring the muscle cells for SK channels and synapse formation in the presence of growing spinal neurons and/or media conditioned by neurons. These studies will enhance our knowledge about how ion channels function, how the channel properties affect the functioning of excitable cells, and give insights about how neurons can regulate the expression of ion channels.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Physiology Study Section (PHY)
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University of Texas Sw Medical Center Dallas
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United States
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