Deficiencies in Na+/H+ exchange compromise fluid and electrolyte transport in the mammalian intestine. In addition, nutrient absorption via H+-coupled peptide transporters and Na+-glucose co-transporters is pH dependent, and may depend upon intracellular pH regulation through Na+/H + exchange in order to remain active. Although a great deal is known about the structure and function of several Na+/H+ exchanger proteins, to date little is known about the remaining family members. In this proposal, we intend to develop a physiologically simple model system where the role of individual Na+/H+ exchangers in mediating intestinal function can be assessed on a genome-wide level in an intact animal. Towards this end, we have cloned the cDNAs for nine Na+/H+ exchangers from the nematode C. elegans.
Aim 1 of this proposal involves determining the cellular, subcellular and temporal expression patterns of these exchangers.
Aim 2 is designed to directly test the physiological role of each exchanger in vivo using double-stranded RNA-mediated gene interference in combination with a novel system to measure intracellular pH in an intact nematode, using GFP as a targetable, non-invasive intracellular pH indicator. Preliminary results suggest that nhx-2, an intestinal Na+/H+ exchanger, is involved in regulating the post-embryonic growth of early larval stage nematodes. Thus, Aim 3 is designed to directly assess the role of this exchanger in mediating nutrient absorption. Using a transposon insertion mutant that lacks nhx-2 gene function, we will examine the uptake of nutrients from a bacterial food source and also assess markers of metabolic potential such as oxygen consumption, superoxide production, and ATP/ADP concentrations. This multidisciplinary approach will help to define the biological role of individual Na+/H+ exchangers in the nematode intestine.
| Denton, Jerod; Nehrke, Keith; Yin, Xiaoyan et al. (2006) Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte. Am J Physiol Cell Physiol 290:C1109-18 |
| Nehrke, Keith (2006) Intracellular pH measurements in vivo using green fluorescent protein variants. Methods Mol Biol 351:223-39 |
| Sherman, Teresa; Chernova, Marina N; Clark, Jeffrey S et al. (2005) The abts and sulp families of anion transporters from Caenorhabditis elegans. Am J Physiol Cell Physiol 289:C341-51 |
| Denton, Jerod; Nehrke, Keith; Rutledge, Eric et al. (2004) Alternative splicing of N- and C-termini of a C. elegans ClC channel alters gating and sensitivity to external Cl- and H+. J Physiol 555:97-114 |
| Nehrke, Keith (2003) A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span. J Biol Chem 278:44657-66 |
