Changes of intra- and extracellular pH (pHj & pHo) can markedly affect ion channels neurotransmitter transport and other properties of neurons and glia. Thus, transporters that move H+/HCO3 across the cell membrane can modulate neuronal activity by changing both pHi and pHo. For chemoreceptor neurons in the medullary raphe (MR) and elsewhere an increase in arterial Pco2 lowers pH1, triggering a compensatory increase in ventilation. In these cells, acidbase tranporters probably influence the chemoreceptor response by establishing resting pH1, determining how far pH1 falls during acid-base disturbances, and mediating a subsequent pH1 recovery that would terminate the response. PH1 measurements indicate that a Na+-driven chloride-bicarbonate exchanger is a key pH1 regulator in brain neurons. We recently cloned such a transporter (NDCBE) from human brain, and also cloned an electroneutral Na/HCO3 cotransporter OF (NBCn); we also have a related transporter (NCBE) whose function is unclear. We will focus on the molecular physiology of Na+-driven Cl-HCO3, exchangers (NDCBE and perhaps NCBE), and the role Na+-coupled HCO3 transporters (SCBTs) play in regulating pH1 in MR neurons and-for comparison-hippocampal (HC) neurons. There are four aims: (i) Develop molecular tools, cloning SCBTs present in MR and HC neurons, and generating type-specific antibodie (ii) Localize SCBT mRNA and proteins, emphasizing the MR and HC. We will perform northern blotting, in-situ hyoridization, PCR, western blotting and immunocytochemistry at the light and EM levels. (iii) Elucidate molecular actions of NDCBE and NCBE. We will determine the function of NCBE; use isotopic fluxes to evaluate ClCl and Na-Na exchange by NDCBE (NCBE); use surface-pH measurements to determine whether NDCBE (NCBE) transports C03; determine how acid-base disturbances affect NDCBE (NCBE); explore the basis for the interaction between carbonic anhydrase II (CAII) and the cytoplasmic C terminus of NDCBE (NCBE); determine whether the DIDS (which inhibits NDCBE) and HCO3 interact with transmembrane segments #3, #5 and #12; and generate NDCBE-lNCBE chimeras. (iv) Elucidate the role of SCBTs in pHj regulation of cultured MR vs HC neurons. We will digitally image fluorescent pH-sensitive dyes to explore the pH1 physiology of identified cells, sometimes using ribozyme to reduce expression of specific SCBTs. Afterwards, we will label these neurons with antibodies to SCBTs and other markers proteins, or identify mRNAs using single-cell PCR. The proposed work will clarify the molecular mechanisrn of Na+-driven Cl-HCO3 exchangers, and the role they and related transporters play in neuronal function. The results could have important implications for understanding neural development, epilepsy, control of ventilation, SIDS and ventilatory adaptation during COPD.
Showing the most recent 10 out of 64 publications
