Studies on electrostatic modulation of membrane physiology

Date of Completion

January 2003

Keywords

Biology, Neuroscience|Biology, Cell|Biophysics, General

Degree

Ph.D.

Abstract

We demonstrated the ability of styryl dyes to measure intramembrane potential (VIM) change associated with asymmetrical surface potentials, by showing the changes in VIM of the bilayer membranes calculated from the fluorescence ratios of these dyes in both lipid vesicles and individual N1E-115 neuroblastoma cells, were consistent with a change in surface potential that can be modeled with the Gouy-Chapman-Stern theory. Next, we imaged the changes in VIM with di-8-ANEPPS following activation of the B 2 bradykinin (BK) receptor on N1E-115 cells. The electric field sensed by the dye increased by an amount equivalent to a depolarization of 83 mV. The increase in VIM was blocked by the phospholipase C (PLC) inhibitors U-73122 and neomycin, and was invariably accompanied by a transient rise of [Ca2+]i. The major portion of the increase in V IM was not attributable to change in transmembrane potential, or change in surface potential induced by a decrease in membrane phosphatidylinositol 4,5-bisphosphate (PIP2). It might arise from an increase in dipole potential, as 1,2-diacylglycerol (DAG) molecules are generated from hydrolysis of other phospholipids. Additionally, we found BK produced an inhibition of a M-type voltage-dependent K current (IK(M)), which was also blocked by PLC inhibitors and had similar kinetics as BK modulation of intramembrane potential, suggesting that the change in the local VIM induced by BK might play a role in mediating the IK(M) inhibition. A biochemical model predicted, and experimental data confirmed that BK stimulated a rapid PIP2 synthesis, and this effect preceded PIP2 hydrolysis by PLC. We then used confocal microscopy to follow the translocation of the pleckstrin homology domain of PLCδ1 fused to green fluorescent protein (PHδ1-GFP) in single cells. The timecourse of PHδ1-GFP translocation could be approximately fit by incorporating binding of PIP2 and IP3 to PHδ1-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve the controversy in the literature over whether PHδ1-GFP is more sensitive to changes in PIP2 or IP3; the model shows that the behavior of this fluorescent probe should be very dependent on its intracellular concentration. ^

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