All forms of life rely on biochemical processes and these processes are either accelerated orinhibited according to the concentration of protons (pH) in their immediate vicinity. In thenervous system, pH buffering mechanisms provide a stable pH environment for biochemicalreactions. Volume-averaged estimates of pH reveal only modest fluctuations in cytosolic andinterstitial pH. Yet changes in pH, much like changes in Ca2+, are likely to be spatially non-uniform, and pH microdomains of substantial magnitude may develop close to the membranesacross which acid equivalents flow. As many membrane-associated receptors, transporters, ionchannels and enzymes are pH sensitive, pH-microdomains could have a significant impact onthe fundamental neuronal properties underpinning normal operations of the nervous system.Our long range goal is to understand the influence of pH-microdomains on neuronal processessuch as membrane excitability, neurotransmission and short term synaptic plasticity, and theextent to which near-membrane pH can influence the recovery of neural function after ischemicevents. Our central hypothesis is that, as Ca2+ is ejected across the plasma-membrane,substantially acidic pH-microdomains develop at the cytosolic face of plasma-membrane Ca2+-ATPases (PMCAs) as a result of H+ exchange for Ca2+. The synaptic cleft will also alkalinize asa result of PMCA activity. Technological limitations have prevented investigations into themagnitude of pH microdomains, and their temporal and spatial characteristics. In aninvestigation of pH microdomains at the synapse, we will overcome current limitations bytargeting pH Indicators to the plasma-membrane of pre- and post-synaptic compartments of theDrosophila neuromuscular junction (NMJ), and to the synaptic cleft. This approach requires thecreation of a number of transgenic flies with ratiometric Genetically Encoded pH Indicators(GEpHIs) fused to proteins with well characterized distributions at the NMJ. We also introduce atechnique to trap chemical pH indicators in the synaptic cleft through the introduction of atetracysteine motif to an extracellular loop of the endogenous presynaptic voltage-gated Ca2+-channel. High speed fluorescence imaging techniques will be used to measure changes influorescence during the action potentials which initiate neurotransmission. Changes influorescence will be calibrated to quantify the underlying changes in pH.
The mechanisms that manage acid levels in the nervous system have limitations; and we propose that these limitations are most pronounced close to the membranes of nerve endings where neurotransmission takes place. A failure to adequately control acid levels in these nerve endings can lead to problems in neurotransmission; as many of the processes serving neurotransmission are sensitive to changes in acid levels. We are proposing the use of a novel technology to investigate acid changes near membranes in nerve endings; which may provide insight into the pathology of epilepsy and the ability of the nervous system to recover after a stroke.
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