All forms of life rely on biochemical processes and these processes are either accelerated or inhibited according to the concentration of protons (pH) in their immediate vicinity. In the nervous system, pH buffering mechanisms provide a stable pH environment for biochemical reactions. Volume-averaged estimates of pH reveal only modest fluctuations in cytosolic and interstitial 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 membranes across which acid equivalents flow. As many membrane-associated receptors, transporters, ion channels and enzymes are pH sensitive, pH-microdomains could have a significant impact on the fundamental neuronal properties underpinning normal operations of the nervous system. Our long range goal is to understand the influence of pH-microdomains on neuronal processes such as membrane excitability, neurotransmission and short term synaptic plasticity, and the extent to which near-membrane pH can influence the recovery of neural function after ischemic events. 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 as a result of PMCA activity. Technological limitations have prevented investigations into the magnitude of pH microdomains, and their temporal and spatial characteristics. In an investigation of pH microdomains at the synapse, we will overcome current limitations by targeting pH Indicators to the plasma-membrane of pre- and post-synaptic compartments of the Drosophila neuromuscular junction (NMJ), and to the synaptic cleft. This approach requires the creation 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 a technique to trap chemical pH indicators in the synaptic cleft through the introduction of a tetracysteine motif to an extracellular loop of the endogenous presynaptic voltage-gated Ca2+- channel. High speed fluorescence imaging techniques will be used to measure changes in fluorescence during the action potentials which initiate neurotransmission. Changes in fluorescence 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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