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• Articles by R.M. Davidson

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• Activation of Phospholipase C Increases Intramembrane Electr...

  Activation of Phospholipase C Increases Intramembrane Electric Fields in N1E-115 Neuroblastoma Cells Biophysical Journal, Volume 84, Issue 6, 1 June 2003, Pages 4144-4156 Chang Xu and Leslie M. Loew Abstract We imaged the intramembrane potential (a combination of transmembrane, surface, and dipole potential) on N1E-115 neuroblastoma cells with a voltage-sensitive dye. After activation of the B bradykinin receptor, the electric field sensed by the dye increased by an amount equivalent to a depolarization of 83mV. The increase in intramembrane potential was blocked by the phospholipase C (PLC) inhibitors U-73122 and neomycin, and was invariably accompanied by a transient rise of [Ca]. A depolarized inner surface potential, as the membrane loses negative charges via phosphatidylinositol 4,5-bisphosphate (PIP) hydrolysis, and an increase in the dipole potential, as PIP is hydrolyzed to 1,2-diacylglycerol (DAG), can each account for a small portion of the change in intramembrane potential. The primary contribution to the measured change in intramembrane potential may arise from an increased dipole potential, as DAG molecules are generated from hydrolysis of other phospholipids. We found bradykinin produced an inhibition of a M-type voltage-dependent K current (). This inhibition was also blocked by the PLC inhibitors and had similar kinetics as the bradykinin-induced modulation of intramembrane potential. Our results suggest that the change in the local intramembrane potential induced by bradykinin may play a role in mediating the inhibition. Abstract | Full Text | PDF (242 kb)

• The Effect of Asymmetric Surface Potentials on the Intramemb...

  The Effect of Asymmetric Surface Potentials on the Intramembrane Electric Field Measured with Voltage-Sensitive Dyes Biophysical Journal, Volume 84, Issue 4, 1 April 2003, Pages 2768-2780 Chang Xu and Leslie M. Loew Abstract Ratiometric imaging of styryl potentiometric dyes can be used to measure the potential gradient inside the membrane (intramembrane potential), which is the sum of contributions from transmembrane potential, dipole potential, and the difference in the surface potentials at both sides of the membrane. Here changes in intramembrane potential of the bilayer membranes in two different preparations, lipid vesicles and individual N1E-115 neuroblastoma cells, are calculated from the fluorescence ratios of di-4-ANEPPS and di-8-ANEPPS as a function of divalent cation concentration. In lipid vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) or from a mixture of the negatively charged lipid phosphatidylserine (PS) and PC, di-4-ANEPPS produces similar spectral changes in response to both divalent cation-induced changes in intramembrane potential and transmembrane potential. The changes in potential on addition of divalent cations measured by the fluorescence ratios of di-4-ANEPPS are consistent with a change in surface potential that can be modeled with the Gouy-Chapman-Stern theory. The derived intrinsic 1:1 association constants of Ba and Mg with PC are 1.0 and 0.4M; the intrinsic 1:1 association constants of Ba and Mg with PS are 1.9 and 1.8M. Ratiometric measurements of voltage sensitive dyes also allow monitoring of intramembrane potentials in living cells. In neuroblastoma cells, a tenfold increase of concentration of Ba, Mg, and Ca gives a decrease in intramembrane potential of 22 to 24mV. The observed changes in potential could also be described by Gouy-Chapman theory. A surface charge density of 1 /115Å provides the best fit and the intrinsic 1:1 association constants of Ba, Mg, and Ca with acidic group in the surface are 1.7, 6.1, and 25.3M. Abstract | Full Text | PDF (396 kb)

• Membrane Electric Properties by Combined Patch Clamp and Flu...

  Membrane Electric Properties by Combined Patch Clamp and Fluorescence Ratio Imaging in Single Neurons Biophysical Journal, Volume 74, Issue 1, 1 January 1998, Pages 48-53 Jing Zhang, Robert M. Davidson, Mei-de Wei and Leslie M. Loew Abstract An experimental method has been established to measure the electric properties of a cell membrane by combination of patch clamp and dual-wavelength ratio imaging of a fluorescent potentiometric dye, 1-(3-sulfonatopropyl)-4-[[2-(di--octylamino)-6-naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS). Pairs of fluorescence images from the dye-stained membrane of neuroblastoma N1E-115 cells excited at two wavelengths were initially obtained to calculate ratio images corresponding to the resting transmembrane potential. Subsequently, a whole-cell patch was established and the membrane potential clamped to levels varying from −100 to +60mV; at each voltage, a pair of dual-wavelength images were acquired to develop a calibration of the fluorescence ratio. Using this method, the resting potentials could accurately be measured showing that the differentiated cells were 17mV more polarized than undifferentiated cells. The combination of electrical and optical methods can also follow changes in other membrane electric properties, such as dipole potential, and thus permit a detailed analysis of the membrane electrical properties underlying the voltage regulation of ion channels. Abstract | Full Text | PDF (130 kb)

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Abstract

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells.