Expression of voltage-gated Ca2+ channel subtypes in cultured astrocytes

Abstract

Transient intracellular [Ca2+] increases in astrocytes from influx and/or release from internal stores can release glutamate and thereby modulate synaptic transmission in adjacent neurons. Electrophysiological studies have shown that cultured astrocytes express voltage-dependent Ca2+ channels but their molecular identities have remained unexplored. We therefore performed RT-PCR analysis with primers directed to different voltage-gated Ca2+ channel α1 subunits. In primary cultures of astrocytes, we detected mRNA transcripts for the α1B (N-type), α1C (L-type), α1D (L-type), α1E (R-type), and α1G (T-type), but not α1A (P/Q-type), voltage-gated Ca2+ channels. We then used antibodies against all of the Ca2+ channel subunits to confirm protein expression, via Western blots, and localization by means of immunocytochemistry. In Western blot analysis, we observed immunoreactive bands corresponding to the appropriate α1 subunit proteins. Western blots showed an expression pattern similar to PCR results in that we detected proteins for the α1B (N-type), α1C (L-type), α1D (L-type), α1E (R-type), and α1G (T-type), but not α1A (P/Q-type). Using immunocytochemistry, we observed Ca2+ channel expression for these subunits in punctate clusters on plasmamembrane of GFAP-expressing astrocytes. These results confirm that cultured astrocytes express corresponding proteins to several high- and low-threshold Ca2+ channels but not α1A (P/Q-type). Overall, our data indicate that astrocytes express multiple types of voltage-gated Ca2+ channels, hinting at a complex regulation of Ca2+ homeostasis in glial cells. © 2003 Wiley-Liss, Inc.