Received for publication April 5, 2006.
Revised May 20, 2006.
Accepted for publication May 23, 2006.

CAPACITATIVE AND OAG-ACTIVATED Ca²⁺ ENTRY DISTINGUISHED USING ADENYLYL CYCLASE TYPE 8

Abstract

Although the molecular identity of capacitative Ca²⁺ entry (CCE) channels remains elusive, TRPC channel family members 3, 6 and 7, which are activated by diacylglycerol (DAG), have been put forward as possible candidates. As HEK 293 cells endogenously express these TRP subunits, this cell line is suitable for investigating whether DAG-activated TRP subunits form part of the putative multimeric assemblies that mediate CCE. Adenylyl cyclase type 8 (AC8) is activated by CCE in non-excitable cells, but is not responsive to other forms of Ca²⁺ entry, such as ionophore-mediated or arachidonate-activated entry through the plasma membrane (Fagan et al., 1996; Fagan et al., 1998; Shuttleworth and Thompson, 1999). In this study, we exploited this unique dependence of AC8 on CCE to ask whether the DAG analogue, 1-oleyl-2-acetyl-sn-glycerol (OAG), activates the same subset of Ca²⁺ channels as store depletion, which triggers CCE. In populations of HEK 293 cells, OAG evoked a faster and greater influx of Ca²⁺ than CCE. Both pathways of Ca²⁺ entry could be triggered simultaneously in the same batch of cells, with additive effects. Strikingly, OAG-mediated Ca²⁺ entry, unlike CCE, did not stimulate AC8 activity in populations of cells. In single cells, OAG evoked a highly heterogeneous response, whereas CCE occurred as a smooth and sustained rise in [Ca²⁺]_i. Taken together, our results indicate that, in HEK 293 cells, OAG-activated Ca²⁺2+ entry is distinct from CCE. The inability of the OAG-activated Ca²⁺ entry pathway to regulate AC8 further reinforces the absolute dependence of this enzyme on CCE.

Key words: Adenylyl cyclases, Calcium (G Protein Coupled Signals)

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