African Journal of Biotechnology Vol. 5 (13), pp. 1303-1307, 3 July 2006   

ISSN 1684–5315 © 2006 Academic Journals        

Full Length Research Paper

The use of confocal microscopy in quantifying changes in membrane potential

Dale Elgar¹*, Fons Verdonck², Anne Grobler³, Carla Fourie¹, Johan Du Plessis¹

¹School for Physiology, Nutrition and Consumer Sciences, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa.

²Interdisciplinary Research Center (IRC), KULAK, B-8500 Kortrijk, Belgium.

³School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa.

*Corresponding author. E-mail: E-mail: fmsde@puk.ac.za; Tel: +27 18 299 2251. Fax: +27 18 299 2248.

Abbreviations: Fluorescence_intra, intracellular fluorescent intensity; fluorescence_intra, extracellular fluorescent intensity; [fluorophores]_intra, intracellular fluorophores concentration; [fluorophores]_extra, extracellular fluorophores concentration; [K_o], extracellular potassium concentration; [K_i], intracellular potassium concentration; MP, membrane potential; OP1, opistoporin 1; PP – parabutoproin; and TMRM, tetramethylrhodamine  methylester.

Accepted 18 May, 2006

Monitoring the plasma membrane potential and its changes can be a time consuming and challenging task especially when conventional electrophysiological techniques are used. The use of potentiometric fluorophores, namely tetramethylrhodamine methylester (TMRM), and digital imaging devices (laser scanning confocal microscopy) provides reliable and time efficient method. Two scorpion pore-forming peptides, namely PP and OP1, were used as a tool to induce depolarization of the plasma membrane potential of neuroblastoma cell line and cardiac myocytes. Alternative methods for the neuroblastoma cells and cardiac myocytes were used. Depolarization of the neuroblastoma cells was calibrated with 140 mM KCl solution with 1 µM valinomycin, after which intensity readers were substituted in the Nernst equation for quantification. Calibration of the alternative method used of the cardiac myocytes’ plasma membrane potential changes was calibrated with the use of 5, 20, 40, and 80 mM KCl solutions with 1 µM valinomycin. A calibration curve was then constructed from which plasma membrane potential could be calculated.

Key words: Membrane potential, TMRM, potassium concentrations, confocal microscopy, parabutoporin, opistoporin1.