This proposal investigates gustatory signal transduction mechanisms using an independently devised dissociation and recording procedure that allows patch clamp analysis of mammalian taste receptor cells. The experiments center on how stimuli representative of sweet and salty taste qualities are transduced to changes of the cell's membrane potential. Sweet taste transduction is thought to be initiated by a cell-surface receptor G-protein mediated stimulation of adenylate cyclase. the consequent elevated cyclic-AMP levels then affect the membrane potential possibly through phosphorylation of potassium channels. Each step in this cascade will be independently tested. The effect of sucrose stimulation (restricted to the apical membrane) on the whole-cell ionic conductances will be examined. Modulation of G-protein function will be tested through GTP-analogues and bacterial toxins. Application of membrane permeant cyclic-AMP analogues or adenylate cyclase activators will test whether sucrose effects and elevated second messenger levels are correlated within the same cell. Phosphorylation will be investigated with activators or inhibitors of protein kinase-A. Single channel experiments are designed to identify the classes of ion channels subserving the change in membrane potential. These channels probably include types of potassium channels and/or channels directly gated by cyclic-AMP. Salt taste transduction studies focus on the seminal role of amiloride-sensitive (AS-) sodium channels in changing the membrane potential. The AS-current will be characterized in detail. Voltage characteristics, amiloride-dose response relations, amiloride analogues, and ion selectivity will be examined. Further, the putative modulation of the AS-current by aldosterone, previously suggested by chorda tympani experiments, will be tested. Another class of sodium currents, the voltage-sensitive (VS-) sodium currents that are essential to the production of action potentials, will be characterized in detail. Properties of the VS-sodium currents may be modulated by activity of AS- sodium currents via increases in intracellular sodium. Single channel experiments are planned to characterize in biophysical detail the AS- sodium channel in terms of dwell times, current-voltage relationship, ion selectivity, and channel conductance. In total, these experiments will help to elucidate the early cellular mechanisms used by taste cells to recognize and transduce stimuli representing sweet and salty to neural events that are ultimately manifest as conscious perception.
