Analysis of growth factor-induced calcium channel upregulation in the PC12 neuronal model system

Melony J Black, Purdue University

Abstract

Modulation of calcium channel expression and/or function in the context of neurotrophin induced neuronal differentiation is still incompletely understood at a mechanistic level. I addressed this issue in the PC12 model neuronal system using patch clamp electrophysiology combined with ectopic expression of human βDGF receptor (βPDGF-R), a surrogate neurotrophin receptor system. PC12 cells ectopically expressing βPDGF-R were treated with PDGF or NGF for up to seven days, and Ca2+ channel subtype expression was analyzed using selective pharmacological agents in both whole cell and cell-attached single channel patch clamp configurations. PDGF-induced upregulation of N and P/Q Ca2+ channel currents completely mimicked upregulation of these currents caused by NGF stimulation of the endogenous TrkA receptor tyrosine kinase (RTK). Neither PDGF nor NGF significantly altered L or R-type currents. Single channel recordings together with immunocytochemistry indicate that growth factor induced increases in whole cell Ca2+ currents were a result of synthesis of new channels. These same data suggested that while increased N channel density was apparent in the soma, additional P/Q channels preferentially distributed to extrasomal locations, probably the proximal neurites. I also observed both NGF and PDGF induced shifts in the steady-state inactivation properties of Ca2+ channels that may reflect changes in expression levels of Ca2+ channel auxiliary γ 2 subunits. Finally, I have begun to investigate the signal transduction events that couple with Ras activation to produce growth factor regulation of Ca2+ channel expression. Signaling deficient mutant forms of βPDGF-R were used to show that activation of Src, PI3-K, RasGAP, PLCγ or Shp2 by RTKs is not required for growth factor induced Ca2+ channel upregulation. In contrast, activation of the Ras-related G-protein Rap1 was found to be critical to this process, however Ras and Rap1 activation together were not sufficient to produce Ca2+ channel upregulation.

Degree

Ph.D.

Advisors

Rane, Purdue University.

Subject Area

Neurology

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