| Summary: | PHARMACOLOGY
Ca2+-SELECTIVE TRPM CHANNELS REGULATE IP3-DEPENDENT Ca2+ OSCILLATIONS IN THE C. ELEGANS INTESTINE
Juan Xing
Dissertation under the direction of Professor Kevin Strange
Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45¡V50 s and mediates defecation. pBoc is controlled by inositol-1,4,5-trisphosphate (IP3)¡Vdependent Ca2+ oscillations in the intestinal epithelial cells. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express an outwardly rectifying Ca2+ (ORCa) current (IORCa) with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL-l and GON-2 function inhibits IORCa 70% and 90%, respectively. IORCa is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating IORCa are interdependent. IORCa, IGON-2, and IGTL-1 have nearly identical biophys+ical properties. Importantly, all three channels are at least 60-fold more permeable to Ca2+ than Na+.
Epistasis analysis suggests that GON-2 and GTL-1 function in a common signaling pathway with PLC× and IP3 receptors to regulate intestinal Ca2+ oscillations. PLC× via hydrolysis of PI(4,5)P2 (PIP2) regulates GON-2/GTL-1 function. Knockdown of PLC× by RNA interference (RNAi) inhibits channel activity ~80%. Inhibition is fully reversed by agents that deplete PIP2 levels. PIP2 added to the patch pipette has no effect on channel activity in PLC× RNAi cells. However, in control cells, 10 ÝM PIP2 inhibits whole cell current ~80%. Channel inhibition by phospholipids is selective for PIP2 with an IC50 value of 2.6 ÝM. Elevated PIP2 levels have no effect on channel voltage and Ca2+ sensitivity and likely inhibit by reducing channel open probability, single channel conductance and/or trafficking. We conclude that hydrolysis of PIP2 by PLC× functions in the activation of both the IP3 receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca2+ influx pathway. Calcium influx through GON-2/GTL-1 feedback regulates its activity and likely functions to modulate IP3 receptor function. PIP2-dependent regulation of GON-2/GTL-1 function may provide a mechanism to coordinate plasma membrane calcium influx with PLC£^ and IP3 receptor activity as well as intracellular Ca2+ store depletion.
Approved: ____________________________________ Date: ______________
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