REGULATION OF AQUAPORIN-1 ION CHANNEL FUNCTION BY INTRACELLULAR SIGNALING PATHWAYS

Abstract

My dissertation work has focused on identifying regulatory mechanisms that govern Cyclic Guanosine Monophosphate (cGMP)-activation of Aquaprorin-1 (Aqp1) ion channels. Aquaporins serve as pores for water thus allowing enhanced water permeability in biological membranes (Preston, et al., 1992). A subset of Aquaporin proteins behave as ion channels regulated by intracellular signaling pathways (Anthony, et al., 2000; Ehring, et al., 1990; Yasui, et al., 1999a). cGMP is necessary for Aqp1 ion channel activation, but only a small subpopulation of Aqp1 proteins function as cGMP-activated ion channels. This observation indicates the involvement of additional regulatory mechanisms in the gating Aqp1 ion channels. Work from this dissertation provides the first insight into the potential mechanism that dictates Aqp1 ion channel availability to respond to the cGMP signal. I show here that insulin-activated tyrosine kinases positively regulate cGMP-mediated activation of Aqp1 ion channels when expressed in Xenopus oocytes. In addition, I show that Aqp1 currents have rapid and slow gating modes with distinct current properties which may reflect distinct gating mechanisms. Treatment of Aqp1-expressing oocytes with insulin enhanced the Aqp1 ion channel function but did not influence the relative frequency of the two gating modes. The Carboxyl (C) -terminus of Aqp1 encodes a PSD-95/DLG/ZO-1 (PDZ) ligand binding domain and a number of putative regulatory domains. The potential regulatory domains were systematically truncated to test the contribution of each of these regions for ion channel availability and current gating. Through this approach I have demonstrated that the enhancement of Aqp1 ion channel by insulin treatment was not mediated by regions of the distal C terminus. I also showed that the regulatory regions of the putative cGMP-binding domain are necessary for the slow but not the rapid gating mode. In summary, results from this dissertation support a hypothesis that Aqp1 is targeted convergently by intracellular signaling pathways which dictate the ion channel status of Aqp1. Work from this dissertation provides evidence that Aqp1 is phosphorylated by an endogenous serine/threonine kinase native to the oocyte. Tyrosine kinases and other signals may serve as 'master switches' governing Aqp1's ability to behave as an ion channel.