| Neurons are specialized cells that can communicate with each other in a high specificity through synapses and be modulated by a large number of extrinsic and intrinsic substances. These functions known as synaptic transmission and cellular excitability are believed to be produced by distinct membrane receptors that selectively bind to specific ligand molecules and couple them to corresponding cellular functions. How the numerous cellular functions are produced by the limited number of receptors, and how these receptors integrate various intra- and extracellular signals are questions that have been asked for years. To test the hypothesis that the G-protein-coupled inward rectifier K+ (GIRK) channel is an integrator that detects the intra- and extracellular signals and couples them to cellular activity, a series of studies on several GIRK channels was carried out in a heterologous expression system Xenopus oocytes using site-direct mutagenesis and electrophysiology approaches. (1) The first question asked was how the GIRK channels receive signals from excitatory neurotransmitters that have been shown to inhibit GIRK in several types of neurons. To prove that the PKC phosphorylation underlies the effect, alanine scanning experiments were performed and a serine residue in the cytosolic domain was identified. Mutation of the PKC Phosphorylation site eliminated the inhibition of GIRK channels by substance P, suggesting that PKC phosphorylation mediates the inhibition of GIRK channels by excitatory neurotransmitters. (2) Since both inhibitory and excitatory neurotransmitters can act on GIRK channels to regulate cellular excitability, the second question asked was how the signals converge onto GIRK channels. The current studies demonstrated a crosstalk between PKC-dependent inhibition and Gbetagamma-dependent activation of GIRK channels, suggesting that PKC-dependent phosphorylation of GIRK channels may function as a molecular switch in mediating the effects of various neurotransmitters. (3) It is possible that the GIRK channels are also modulated by metabolic substrates and products in addition to hormones and neurotransmitters. Therefore the third question asked was whether GIRK channels can integrate signals of extracellular pH changes, as pH fluctuations are seen in a number of physiologic and pathophysiologic conditions. These studies indicated that GIRK channels were reversibly activated by extracellular protons via augmentation of single-channel conductance through protonation of a histidine in the extracellular domain, implying that protons contribute to the GIRK-mediated synaptic transmission. (4) If the GIRK channels are stimulated by extracellular protons, how does intracellular pH affect the channels? Thus experiments were conducted to answer this question. It was found that selective intracellular acidification inhibited GIRK channel by decreasing the channel open-state probability. Three histidines were identified to be responsible for the proton-induced inhibition of GIRK channels. Because of the inequality of pHi and pH o in most cells and their relatively independent controls by cellular versus systemic mechanisms, the differential modulation of GIRK channel by pHi and pHo may allow GIRK channels to regulate cellular excitability in certain physiological and pathophysiological conditions. (Abstract shortened by UMI.)... |
