The parathyroid (PT) cell plays a critical role in mineral ion homeostasis by sensing small changes in the extracellular calcium concentration ([Ca2+]omicron) and responding with oppositely directed changes in parathyroid hormone (PTH) secretion. Existing evidence suggests that PT cells recognize and respond to (e.g., """"""""sense"""""""") changes in [Ca2+] omicron through a cell surface, """"""""receptor-like"""""""" mechanism, that is coupled to intracellular (IC) effector systems and hormonal release via one or more guanine-nucleotide regulatory (G)-proteins. Much of this evidence is indirect, reflecting a lack of direct methods for identifying and characterizing the putative """"""""Ca2+-receptor"""""""" and its coupling to more distal biological responses. Recently, novel techniques have been developed which permit detailed analysis of the transduction mechanisms, including the role of G-proteins and second messengers, coupling cell surface receptors to their respective IC effector systems at the level of the single cell. Among the most powerful of these utilize the patch clamp technology and permit control of the IC milieu with simultaneous monitoring of parameters relevant to secretory physiology, including the activity of plasma membrane ion channels, membrane voltage (V/m), and the cytosolic Ca2+ concentration ([Ca2+]i). The overall goal of this proposal is to employ this methodology to define the mechanisms coupling the [Ca2+]omicron signal to the control of [Ca2+]i, (V/m, and plasma membrane ion channels in PT cells. This information is an essential foundation for further dissection of the components required for Ca2+-sensing and their eventual reconstitution using purely in vitro systems. The project will address the following questions: (1) Do the high [Ca2+]omicron-elicited [Ca2+]i transients in PT cells result from the generation of inositol 1,4,5-triphosphate by direct, G-protein-mediated activation of phospholipase C (PLC? (2) What are the plasma membrane Ca2+ influx pathways activated at high [Ca2+]omicron in PT cells, and how are they regulated by (V/m), G-proteins, and second messengers? (3) Does [Ca2+]omicron control (V/m) in PT cells by a G-protein, and/or second messenger-dependent mechanism, and what are the ion channel(s) involved in this regulation? (4) At the single channel level, what are the roles of G-proteins and second messengers in coupling the [Ca2+]omicron signal to the regulation of a large conductance K+ channel in PT cells? These studies should provide further insight into the mechanisms through which the [Ca2+]omicron signal coupled to intracellular effector systems not only in PT cells but also in other cells sensing [Ca2+]omicron.
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