Evidence for extracellular calcium-sensing receptor mediated opening of an outward K+ channel in a human astrocytoma cell line (U87).

Abstract

An extracellular calcium (Ca2+o)-sensing receptor (CaR) plays crucial roles in maintaining systemic calcium homeostasis. The CaR is also expressed in other cells uninvolved in systemic mineral ion homeostasis, including keratinocytes, fibroblasts, and neurons. In brain the CaR is widely distributed, being particularly abundant in neurons in subfornical organ, cingulate cortex, hippocampus, and cerebellum. It is also present in fiber tracts in rat brain, presumably in oligodendroglia and in cultured rat oligodendrocytes, suggesting that the CaR modulates the function of nonneuronal cells within brain. In this report, we show functional CaR expression in a human astrocytoma cell line (U87). Reverse transcription-polymerase chain reaction (RT-PCR) amplified a product from U87 cell RNA exhibiting >98% homology with the human CaR. Northern blot revealed a 5.5 kb transcript, similar to the principal transcript in human parathyroid, and a smaller 2.4 kb transcript. U87 cells expressed CaR protein as assessed by immunocytochemistry and Western blot using an affinity-purified, anti-CaR antiserum. Patch clamp analysis in the cell-attached mode revealed that raising Ca2+o from 0.75 to 1.75 or 2.75 mM produced approximately threefold increases in the open state probability (Po) of an outward K+ channel with a conductance of approximately 88 pS. A specific "calcimimetic" CaR activator, R-467 (0.5 microM), activated this K+ channel similarly, while its less active stereoisomer, S-467, did not. Thus U87 astrocytoma cells express both CaR mRNA and protein, and the receptor activates an outward K+ channel previously suggested to be involved in membrane polarization and cellular excitability.