Ion channels are large protein macromolecules which span cell membranes. They open and close, or gate their pores, controlling the flux of ions across the membrane, and consequently, membrane potential. The long term objectives of the proposed research are to determine the gating mechanisms of ion channels. To work towards this goal, currents will be recorded from single ion channels with the patch clamp technique and analyzed by computer. The channels to be studied are the large conductance calcium-activated potassium channel (BK channel) and the fast Cl channel, obtained from the membrane of mammalian skeletal muscle cells grown in tissue culture. Seven specific projects will be carried out: (1) to determine whether the gating kinetics of ion channels are best described by models with discrete states and constant transition rates between the states (Markovian models) or by models with a continuum of states and fractal scaling (fractal models); (2) to determine whether the brief interruptions (flickers) commonly observed in currents flowing through single channels arise from complete or partial channel closures; (3) to implement an advanced method for determining kinetic gating mechanisms, which uses all of the non-redundant kinetic information in the single channel current record and which takes into account both limited time resolution and the noise in the current record. This method uses computer simulation to calculate, for a given gating mechanisms, the two-dimensional distributions of adjacent open and shut interval durations, which are then compared to the experimental distributions. This advanded method will be used to determine: (4) the steady-state gating mechanism of the fast Cl channel; (5) mechanism by which voltage modulates the activity of the fast Cl channel; (6) the Ca-activated gating mechanism for the normal mode of the BK channel; and (7) the altered gating mechanisms for the other modes of the BK channel. In each case, the most likely gating mechanisms will be defined in terms of kinetic schemes which indicate: the numbers of open and shut states, the transition pathways between the states, the energy barriers for the transitions, and how channel activity is modulated through voltage or calcium induced changes in energy barrier heights. Characterizing ion channels is an important step towards understanding the molecular basis of both normal muscle function and those muscle diseases where defects in the numbers and/or functions of ion channels are implicated. Once the normal channels are characterized, it will be possible to determine if their numbers and/or functions are altered in the disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR032805-13
Application #
2078876
Study Section
Special Emphasis Panel (NSS)
Project Start
1983-09-01
Project End
1998-08-31
Budget Start
1995-09-01
Budget End
1996-08-31
Support Year
13
Fiscal Year
1995
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
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
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
Shelley, Christopher; Niu, Xiaowei; Geng, Yanyan et al. (2010) Coupling and cooperativity in voltage activation of a limited-state BK channel gating in saturating Ca2+. J Gen Physiol 135:461-80
Shelley, Christopher; Magleby, Karl L (2008) Linking exponential components to kinetic states in Markov models for single-channel gating. J Gen Physiol 132:295-312

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