Cloning, expression and inhibitor studies of organellar Ca2-ATPases

Lockyer, Peter John (1997) Cloning, expression and inhibitor studies of organellar Ca2-ATPases. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

A complementary DNA for the Tobacco Budworm, Heliothis virescens, sarco (endo)plasmic reticulum-type Ca²⁺-ATPase (HVSERCA) has been cloned and sequenced. The cDNA was cloned from a random-primed cDNA library made from 4^th and 5^th instar larvae polyA⁺ RNA. cDNA fragments of adult rabbit fast-twitch muscle CVa²⁺-ATPase (SERCA1a) were used as heterologous probes to isolate a partial cDNA clone which coded for a protein with high amino acid homology to the Ca²⁺-ATPase of Drosophila melanogaster (DRSERCA) and vertebrate ER/SR calcium pumps. Fragments of this Heliothis clone were utilised as probes to 'walk' upstream. The extreme 5' end of the insect Ca²⁺-ATPase cDNA sequence was isolated by PCR. The entire cDNA clone contains an open reading frame of 1000 codons and shares the characteristic motifs of P-type ATPases. The five nucleotides upstream of the start codon (CCACCAUGG) share the same sequence as the consensus Kozak transitional start sequence. It is the third arthropod ER/SR CA²⁺-ATPase to be cloned and has high amino acid identity with DRSERCA and about 70% identity with mammalian SERCAs.

Two novel inhibitors of rabbit SERCA1a enzyme have been identified, ellagic acid and curcumin. ATPase activity of purified rabbit skeletal SR is inhibited by ellagic acid and curcumin at an IC₅₀ of 10 μM and 4 μM, respectively. Ellagic acid raises [Ca²⁺]_i in Quail fibroblasts imaged by confocal microscopy with the Ca²⁺ dye Indo-1. In contrast, curcumin does not raise [Ca²⁺]_i, which would be expected for a Ca²⁺-ATPase inhibitor, and prevents an elevation of [Ca²⁺]_i by the potent Ca²⁺ pump inhibitors, 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) and trilobilide. This effect may provide a mechanism for the role of curcumin as an anti-cancer agent.