Doctor of Philosophy (PhD)
Nitric Oxide (NO) is a key gaseous messenger that has been shown to be produced by multiple cell types in the vertebrate retina. Research in our lab is aimed at unlocking critical synaptic functions of NO. A major finding from our lab is that NO affects synaptic responses in amacrine cells by altering the plasma membrane gradient for Cl-. This is due to release of Cl- from an internal store and this in turn dependent on a decrease in cytosolic pH. Determining the factors regulating cytosolic Cl- in neurons is fundamental to our understanding of the function of GABAergic and glycinergic synapses. This is because the Cl- distribution across the postsynaptic plasma membrane determines the sign and strength of postsynaptic voltage responses. Here, my goals were to confirm the compartmental nature of the internal Cl- store and to test the hypothesis that Cl- is being released from acidic organelles such as the Golgi, synaptic vesicles, endosomes or lysosomes. To accomplish this, I made whole cell voltage clamp recordings from cultured chick retinal amacrine cells and used GABA-gated currents to track changes in cytosolic Cl-. The compartmental pH was monitored using LysoSensor imaging. My results demonstrate that increasing compartmental pH with internal weak bases leads to release of Cl- into the cytosol and subsequent addition of NO causes a reduction rather than the usual increase in cytosolic Cl-. In contrast, collapsing proton gradients and thus proton-dependent membrane potentials completely blocked the ability of NO to release compartmental Cl-. These results indicate that maintenance of internal proton gradients is critical to the mechanisms of Cl- release and that Cl- is likely to come from acidic organelles. Additionally, I tested the hypothesis that the store can be emptied and refilled. I determined the conditions under which the store can be depleted and the dependence of refilling on extracellular Cl-. These results demonstrate that the regulation of cytosolic Cl- is closely linked to multiple regulatory processes and further our understanding of repertoire of NO signaling mechanisms.
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Krishnan, Vijai, "Regulation of the Chloride Store in the Retinal Amacrine Cells" (2015). LSU Doctoral Dissertations. 2825.