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

Abstract

Transient intracellular [Ca(2+)] 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 Ca(2+) channels but their molecular identities have remained unexplored. We therefore performed RT-PCR analysis with primers directed to different voltage-gated Ca(2+) channel alpha(1) subunits. In primary cultures of astrocytes, we detected mRNA transcripts for the alpha(1B) (N-type), alpha(1C) (L-type), alpha(1D) (L-type), alpha(1E) (R-type), and alpha(1G) (T-type), but not alpha(1A) (P/Q-type), voltage-gated Ca(2+) channels. We then used antibodies against all of the Ca(2+) 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 alpha(1) subunit proteins. Western blots showed an expression pattern similar to PCR results in that we detected proteins for the alpha(1B) (N-type), alpha(1C) (L-type), alpha(1D) (L-type), alpha(1E) (R-type), and alpha(1G) (T-type), but not alpha(1A) (P/Q-type). Using immunocytochemistry, we observed Ca(2+) channel expression for these subunits in punctate clusters on plasma membrane of GFAP-expressing astrocytes. These results confirm that cultured astrocytes express corresponding proteins to several high- and low-threshold Ca(2+) channels but not alpha(1A) (P/Q-type). Overall, our data indicate that astrocytes express multiple types of voltage-gated Ca(2+) channels, hinting at a complex regulation of Ca(2+) homeostasis in glial cells.