Ion channels are proteins that control the passive flux of selected ions through cell membranes by opening and closing (gating) their pores. Ion channels play a key role in many physiological processes, including integration of information in neurons, propagation of action potentials in nerve and muscle cells, synaptic transmission, and control of hormone secretion. This proposal seeks to continue work on investigating the fundamental mechanisms by which ion channels gate their pores. The focus of this research is on large conductance Ca2+-activated K+ (BK) channels from nerve and muscle. Although it is known that BK channels are activated by both increases in internal Ca2+ and depolarization of the membrane potential, the mechanisms by which this activation occurs is not clear. To work towards answering this question, the patch-clamp technique will be used to record ionic currents flowing through single BK channels in cultured rat skeletal muscle and also from cloned BK channels expressed in Xenopus oocytes and the HEK (human embryonic kidney) 293 cell line. The single-channel currents will then be analyzed with state-of-the-art techniques to determine the kinetic gating mechanisms of the channels. Such gating mechanisms will specify the numbers of states entered during gating, the transition pathways among the states, the rate constants for the transitions, and the changes in the rate constants produced by Ca2+i, voltage, and the beta subunit of the BK channel.
Four specific aims will be carried out to resolve: 1) the mechanism by which the channels gate over the range of very low to very high Ca2+i; 2) the contributions of proposed primary and secondary Ca2+-binding sites to the gating; 3) the mechanism by which voltage activates the channels; and 4) the mechanism by which the beta subunit increases the Ca2+i sensitivity and alters the gating. An important step towards understanding how ion channels gate their pores and determining the contributions of ion channels to normal cellular function, as well as to identifying defective channels in disease processes, is to establish the kinetic gating mechanisms of the channels. This proposal will work towards this goal for BK channels.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR032805-16
Application #
2692034
Study Section
Physiology Study Section (PHY)
Project Start
1983-09-01
Project End
2003-08-31
Budget Start
1998-09-01
Budget End
1999-08-31
Support Year
16
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
Geng, Yanyan; Magleby, Karl L (2015) Modal gating of endplate acetylcholine receptors: A proposed mechanism. J Gen Physiol 146:435-9
Geng, Yanyan; Wang, Xiaoyu; Magleby, Karl L (2013) Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers. J Gen Physiol 141:493-7
Landowne, David; Yuan, Bin; Magleby, Karl L (2013) Exponential sum-fitting of dwell-time distributions without specifying starting parameters. Biophys J 104:2383-91
Budelli, Gonzalo; Geng, Yanyan; Butler, Alice et al. (2013) Properties of Slo1 K+ channels with and without the gating ring. Proc Natl Acad Sci U S A 110:16657-62
Fernandez, Jose A; Skryma, Roman; Bidaux, Gabriel et al. (2012) Short isoforms of the cold receptor TRPM8 inhibit channel gating by mimicking heat action rather than chemical inhibitors. J Biol Chem 287:2963-70
Geng, Yanyan; Niu, Xiaowei; Magleby, Karl L (2011) Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume. J Gen Physiol 137:533-48
Manzanares, Dahis; Gonzalez, Carlos; Ivonnet, Pedro et al. (2011) Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume. J Biol Chem 286:19830-9
Chen, Ren-Shiang; Geng, Yanyan; Magleby, Karl L (2011) Mg(2+) binding to open and closed states can activate BK channels provided that the voltage sensors are elevated. J Gen Physiol 138:593-607
Holohean, Alice M; Magleby, Karl L (2011) The number of components of enhancement contributing to short-term synaptic plasticity at the neuromuscular synapse during patterned nerve Stimulation progressively decreases as basal release probability is increased from low to normal levels by changing J Neurosci 31:7060-72
Fernandez, Jose A; Skryma, Roman; Bidaux, Gabriel et al. (2011) Voltage- and cold-dependent gating of single TRPM8 ion channels. J Gen Physiol 137:173-95

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