| Studies of central CO2 chemosensors are hampered by the complexity of mammalian central nervous systems. Our laboratory developed the pulmonate snail, Helix aspersa, as a relatively simple alternative to mammalian models. A question concerning central CO2 chemosensor function is the mechanism by which central CO2 chemosensors regulate pHi. A finding common to mammals and H. aspersa is that putative central CO2 chemosensors express relatively poor pHi regulation. The basis for poor pHi regulation has been explained by expression of an NHE activity that is sensitive to inhibition by extracellular protons. This thesis research was directed at determining how extracellular protons inhibit H. aspersa NHE activity. Our hypothesis was that histidines mediate the pHe sensitivity of H. apsersa NHE1.;To test this hypothesis, NHE activity in H. aspersa neurons was measured during hypercapnia and isohydric hypercapnia using the fluorescent pH indicator, BCECF. pHi regulation differed among H. aspersa neurons and a pHe sensitive NHE activity was expressed by some neurons. H. aspersa NHE1 cDNA was next cloned from the snail's central nervous system using a degenerate PCR approach. Sequence analysis identified several histidines with the potential to act as pHe sensors. By single cell PCR, it was found that H. aspersa NHE1 was expressed by all neurons but to varying degree. We were ultimately unable to express H. aspersa NHE1 protein. Thus, the role of histidines in mediating the pHe sensitivity of rat NHE1 was tested and related to H. aspersa NHE1 through sequence comparison. Rat NHE1 histidines were mutated to alanin individually, and some in pairs, by site-directed mutagenesis. The effect on NHE1 protein was assayed by measurement of pHi regulatory activity using BCECF. Two histidines were found to mediate pHe sensitivity, H84 and H279. Interestingly, H84 inhibited but H279 facilitated NHE1 activity. There is no H. aspersa analog of H84 but the counterpart to H279 is an arginine residue, R193. Arginine is positively charged at physiologic pH and is known to be involved in the pH sensitivity of proteins. We conclude that R193 is a good candidate for a pHe sensor in H. aspersa NHE1. |
