MECHANISMS OF IONIC CHANNEL ACTIVITY

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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.