The goals of the proposed research are twofold: first, to characterize and identify the negative fixed charges which line the external surface of myocardial cells and are functionally associated with gating and ion permeation of membrane channels; and second, to determine how these moieties might influence the actions of organic calcium antagonists. To achieve these goals we will (a) determine the surface charge density associated with calcium channels, """"""""delayed rectifier"""""""" potassium channels, and """"""""hyperpolarization-activated"""""""" channels in single mammalian myocytes; (b) test the hypothesis that phospholipids and sialic acids constitute much of the """"""""functionally relevant"""""""" surface charge; (c) determine how altering the surface potential might influence tonic, use-dependent and voltage-dependent actions of organic calcium antagonists; and (d) test the hypothesis that negative surface charge derived specifically from phospholipids and sialic acids plays and important role in modulating these actions. A single microelectrode voltage clamp technique will be employed to obtain steady state activation/inactivation-voltage curves for each type of channel; shifts in these curves, produced by multivalent cations, will be used to calculate surface charge densities. Similar measurements will be made after cells have been pretreated with phospholipases or neuraminidase (to remove sialic acid). Then the enzyme-induced changes in surface charge density will be correlated with alterations in the membrane phospholipid distribution pattern or sialic acid content. The steady state inactivation curve for the calcium channel will be determined before and after addition of D600 or nisoldipine, and established pulse protocols will be used to characterize their actions before and after addition or removal of divalent cations, and after cells have been pretreated with phospholipase or neuraminidase. These studies are important because surface potentials modulate gating and ion permeation of membrane channels, functions that are critical to the contractile performance and pacemaker activity of the heart. The work is clinically relevant since the surface potential can presumably affect the binding and actions of organic calcium antagonists, or can be altered, itself, by pH, hyper or hypocalcemia, and by antiarrhythmic drugs. Identifying the molecular substrates of the surface potential is important not only for future characterization of ion channel structure-function relationships, but also for possible clinical use of this information.
