Different effects of Hg²⁺ and Cu²⁺ on mussel (Mytilus galloprovincialis) plasma membrane Ca²⁺-ATPase: Hg²⁺ induction of protein expression

Deregulation of Ca²⁺ homeostasis can produce serious effects on cell functioning due to an alteration of Ca²⁺ signaling. The aim of this study was to evaluate variations in plasma membrane Ca²⁺-ATPase (PMCA) induced in mussels by in vivo exposure to Cu²⁺ or Hg ²⁺. PMCA activity was assayed using a cytochemical method allowing localization and in situ quantification of Ca²⁺-ATPase on cryostat tissue sections. The effects of fixed concentrations of Cu²⁺ (0.6 μM) or Hg²⁺ (1.3 μM) were evaluated after different times of exposure (1, 4, 6 days), while those of increasing amounts of Cu²⁺ (0.3, 0.6, 1.3 μM) or of Hg²⁺ (0.6, 1.3, 2.4 μM) were evaluated after 4 days. Cu²⁺ produces dose-dependent inhibition of PMCA in the digestive gland, with a minimum at the fourth day of treatment and a recovery at the sixth day. Conversely, Hg²⁺ induces a significant rise of PMCA activity, with a maximum at the fourth day. Similar results have been found after biochemical assay of PMCA, using plasma membranes obtained from density-gradient separation of gill homogenates. PMCA expression has been assessed by immunoprecipitation and Western immunoblotting on digestive gland homogenates, showing an induction after exposure to Hg²⁺ but not to Cu²⁺. In conclusion, Cu²⁺ does not vary PMCA expression but reduces PMCA activity, indicating PMCA inhibition; conversely, Hg ²⁺ increases PMCA expression more than PMCA activity, suggesting that it also produces PMCA inhibition, but the induction of PMCA expression leads to a net increase in enzyme activity.