The inhibitory effect of Mn 2+ on the ATP-dependent Ca 2+ pump in rat brain synaptic plasma membrane vesicles

Abstract

Synaptic plasma membrane (SPM) vesicles take up Ca 2+ when both ATP and Mg 2+ are added to the reaction medium. Maximal ATP-dependent Ca 2+ uptake is obtained with between 3 and 5mM Mg 2+. Higher [Mg 2+] results in a slight decrease in the ATP-dependent Ca 2+ influx, which at 10 mM Mg 2+ is equal to 70% of the maximal uptake. [Mn 2+] up to 0.3 mM, Ca 2+n support ATP-dependent Ca 2+ uptake as efficiently as equimolar [Mg 2+]. Maximal ATP-dependent Ca 2+ uptake in the presence of Mn 2+ is obtained between 1 and 2 mM but its extent is only 70% of the maximal uptake obtained in the presence of Mg 2+. As the [Mn 2+] increases the ATP-dependent Ca 2+ uptake decreases and at 10 mM Mn 2+ it is only 40% of that obtained in the presence of Mg 2+. The reduction in ATP-dependent Ca 2+ uptake in the presence of Mn 2+ is obtained also when Mg 2+ is present in the reaction medium. The effect is independent of the [Mg 2+] used and depends on the [Mn 2+ ] alone. Studying the relationship between [Mn 2+] and [Ca 2+] revealed three patterns: at 0.3 mM Mn 2+ this ion could replace Mg 2+ over the entire [Ca 2+] range tested, 5–200 μM, supporting ATP-dependent Ca 2+ uptake. When the [Mn 2+] was increased to 1.8 mM, it exhibited a competitive behaviour with Ca 2+ which resulted in an increase in apparent K m to Ca 2+ of the SPM Ca 2+ pump from 10.69 μM (SD = 3.49) to 28.88 μM (SD = 21.08). Four millimolar Mn 2+ inhibited ATP-dependent Ca 2+ uptake to 50% of that obtained in equimolar Mg 2+ over the entire [Ca 2+], 5-200 μM, tested. No transport of 54Mn 2+ by the SPM Ca 2+ pump could be detected. Inclusion of Mn 2+ in the reaction medium led to excessive phosphorylation of SPM proteins. The excessive levels of phosphorylation persisted also when Ca 2+ and/or Mg 2+ were present in the reaction media.