Mark O. Bevensee - US grants

DESCRIPTION (provided by applicant): In brain, pH regulation is important because many ion channels, neurotransmitter-uptake systems, and cellular processes are sensitive to changes in pH. To regulate both intracellular pH (pHi) and extracellular Ph (pHo), brain cells such as neurons and glia utilize plasma-membrane transporters to shuttle hydrogen and bases such as bicarbonate across their membranes. Bicarbonate-coupled transporters such as Na/Bicarbonate Cotransporters (NBCs) are particularly potent regulators of brain pH, and electrogenic ones occupy an unusual niche in contributing to activity-evoked changes in pH. Our understanding of this family of proteins has expanded following the molecular identification of many of these proteins. The long-term objective of this proposal is to elucidate the physiological significance of multiple electrogenic NBCs in brain. Antibodies to specific isoforms will be used to examine the expression profiles of the proteins throughout the rat brain (Aim 1). Subsequently, the contribution of each NBC to the pH physiology of the rat hippocampus will be explored. The identification and biophysical characterization of NBC activity in each hippocampal cell type will be revealed and compared using fluorescence imaging of pHi and patch-clamp techniques (Aim 2). Functional properties will therefore be assigned to the molecules identified in Aim 1. Finally, to elucidate the structural features that underlie the functional properties of the NBCe1 proteins studied in Aim 2, structure-function relationships of NBCe1 will be examined (Aim 3). Utilizing available information on related anion exchangers (AEs), specific regions and residues responsible for ion binding and translocation, as well as pH and voltage sensitivities will be determined. Chimeric NBC-AEs and truncated/mutant NBCs will be expressed and functionally characterized in oocytes using microelectrodes and the macropatch technique, as well as in transfected mammalian cells using fluorescence imaging. Studies will involve examining fundamental bicarbonate transport, as well as the more detailed transport properties of ion, pH, and voltage dependencies and activation kinetics. Results will provide information pertinent to other members of the superfamily. Understanding the expression, function, and structure of electrogenic NBCs will help clarify the influence of these proteins on neuronal activity and synaptic transmission, as well as their involvement in acid-base disturbances such as epilepsy, ischemia, and hypoxia.

DESCRIPTION (provided by applicant): Both genetic forms of polycystic kidney disease (PKD) present in human or mouse models as a profound change in renal tubule or epithelial cell morphology and architecture due to mutations in proteins that localize, at least in part, to the apical central monocilium of the cortical collecting duct (CCD) principal cell (PC cell). Once the genetic and biochemical consequences of PKD are manifested in this change in morphology, the change in cellular or tubular architecture affects transepithelial ion transport profoundly. In human autosomal recessive PKD (ARPKD) monolayers, there is evidence of sodium hyperabsorption, although the sodium transport mechanisms are not yet clearly defined. This abnormality may explain early onset hypertension observed in the majority of human ARPKD patients. Using mouse renal epithelial cells that are immortalized due to genetic cross with the Immortomouse and form polarized epithelial cell monolayers from wild-type, mutant, and genetically rescued PC cells from the Oak Ridge polycystic kidney (orpk) mouse CCD of very high electrical resistance, our laboratory has gathered preliminary data showing upregulated absorptive sodium transport in mouse orpk ARPKD mutant cortical collecting duct (CCD) principal epithelial cells (PC cells) grown as polarized monolayers and lacking apical central monocilia versus control cilium-competent PC cell monolayers. These upregulated sodium currents may represent ENaC- and NHE-mediated sodium hyperabsorption. Taken together, the literature, the experience of our collaborative research group, our current preliminary work, and the constructive criticism of the reviewers of our original application led us to formulate the following working hypothesis: CCDs from mouse models of ARPKD that lack apical central monocilia have upregulated ENaC- and NHE-mediated sodium absorption and resultant hypertension. Interrelated specific aims derive from this hypothesis and are designed to understand the cellular and molecular mechanisms that underlie this ARPKD disease phenotype.

The regulation of intracellular pH (pHi) and extracellular pH (pHo) of brain is critical for optimizing neuronal excitability. Na-Coupled Bicarbonate Transporters (NCBTs) ?particularly the astrocytic electrogenic Na/bicarbonate cotransporter NBCe1 that couples changes in pH with neuronal activity? are key regulators of brain pH. Despite the clear importance of pH regulation and the abundance of NBCe1 in brain, the role of NBCe1 and associated pH changes in modulating synaptic transmission and synaptic plasticity has not be identified. The current objective is to use molecular and pharmacological tools with electrophysiological approaches in brain-slice studies to investigate the role of NBCe1 and associated pH changes in modulating hippocampal synaptic function. Aim 1 is to address the hypothesis that NBCe1 dampens basal, low-frequency synaptic transmission. A combination of extracellular and whole-cell recordings in CA1 of acute hippocampal slices from wild-type and NBCe1 knockout (KO) mice will be used to investigate the role of NBCe1 in regulating synaptic transmission and spiking in hippocampal CA1 in response to low-frequency (0.1 Hz) extracellular stimulation. The effects of NBCe1 inhibitors (S0859 and a function blocking L3 antibody), a function stimulating L4 antibody, and viral restoration of NBCe1 into astrocytes in NBCe1 KO mice will be determined. pH-sensitive microelectrodes and dyes will be used to examine associated changes in pHo, as well as the pH of astrocytes and presynaptic terminals to test the hypothesis that inhibiting NBCe1 stimulates basal synaptic transmission by enhancing the extracellular alkaline shift (established conventional model). Aim 2 is to address the hypothesis that NBCe1 enhances high-frequency synaptic transmission and long-term plasticity. The molecular and pharmacological tools and approaches described for Aim 1 will be used to examine excitatory synaptic responses, but in response to high-frequency (50 Hz) extracellular stimulation. pH measurements will be made in the extracellular space, astrocytes, and presynaptic nerve terminals to test the hypothesis that NBCe1 stimulation of high-frequency synaptic transmission and long-term potentiation (LTP) involves dampening of the activity-evoked presynaptic pHi decrease (new mechanism). Underlying mechanisms such as pre- vs postsynaptic responses and the role of specific receptors will be evaluated for both Aims. Results will reveal that NBCe1 is a physiologically important acid-base transporter that modulates synaptic transmission and LTP through changes in pH that are dependent on frequency stimulation. The results will contribute to our understanding of NBCe1 and associated pH changes in neuronal activity, seizures, ischemia, and hypoxia.