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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca²⁺-ATPase

¹Periparturient Diseases of Cattle Research Unit, and ²Bacterial Diseases of Livestock Research Unit, United States Department of Agriculture-Agricultural Research Service, National Animal Disease Center, Ames, Iowa 50010

Submitted 19 February 2003 ; accepted in final form 6 September 2003

On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca²⁺-ATPase (SPCA). Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin. Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker -mannosidase II. To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells. Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector. Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca²⁺-ATPase, sarco(endo)plasmic reticulum Ca²⁺-ATPase, and calreticulin expression in these clones. In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly. The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La³⁺ treatment. Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells. The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector. These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells.

calcium transport

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