Our long-term objectives are: 1. To determine the topographical organization of voltage- and calcium-gated ion channels in the membrane of mammalian nerve cells and gain insight into the manner by which these cells process and transmit information. 2. To understand how this organization is achieved during differentiation and what mechanisms are responsible for its maintenance. 3. To elucidate the role of voltage- and calcium-gated ion channels in the regulation of such key neuronal processes as directed neurite outgrowth and synapse formation. Towards achievement of these objectives we plan to characterize the diverse classes of voltage- and calcium-gated ion channels in the membrane of neuronal cell lines at different stages of growth and differentiation and determine the spatial arrangement of these channels in the different regions of a single cell. We have recently recorded single channel currents from growth cones of differentiated neuroblastoma cells with the cell attached patch clamp technique. We have observed four classes of voltage-gated channel events that could be distinguished by their direction, amplitude and threshold voltage. We plan to expand these studies along a number of lines. 1. Using the cell attached patch clamp technique, we will further examine the distribution of the different types of voltage- and calcium-gated ion channels in the growth cone region, and explore their localization in the neurites and in the soma. We will also examine the distribution of sodium and potassium channels in the different regions of the differentiated cell by recording macroscopic currents locally with the loose patch clamp technique. 2. Using """"""""cell free"""""""" patches from each region in the inside-out or outside-out configurations we will identify characterize the different classes of channels by investigating their selectivity, calcium sensitivity, voltage dependency, pharmacological and kinetic properties. 4. Using cells undergoing rapid and synchronous differentiation in culture, we will determine the sequence of expression of voltage- and calcium-gated ion channels by recording single channel activity and local membrane currents from the different regions of the developing cell.
| Bolsover, S R; Gilbert, S H; Spector, I (1992) Intracellular cyclic AMP produces effects opposite to those of cyclic GMP and calcium on shape and motility of neuroblastoma cells. Cell Motil Cytoskeleton 22:99-116 |
| Bolsover, S R; Spector, I (1986) Measurements of calcium transients in the soma, neurite, and growth cone of single cultured neurons. J Neurosci 6:1934-40 |
