Calcium transport mechanisms in membrane vesicles from guinea pig brain synaptosomes

Ca²⁺ transport mechanisms were investigated using membrane vesicles prepared from guinea pig brain synaptosomes by hypotonic lysis. Two major mechanisms of Ca²⁺ transport exist, Na⁺-Ca²⁺ exchange and ATP-dependent Ca²⁺ uptake. A third although minor component of Ca²⁺ uptake occurs under hyperpolarizing conditions (determined by increased uptake of [³H]tetraphenylphosphonium⁺). Na⁺-Ca²⁺ exchange results in a rapid increase of [Ca²⁺](i) (up to 100-fold above [Ca²⁺]₀), has a K(m) for Ca²⁺ of 40 μM, is fully reversed by added external Na⁺, is inhibited by agents dissipating Na⁺ gradients (monensin or veratridine), and is uninfluenced by mitochondrial inhibitors. ATP-dependent Ca²⁺ uptake has a higher affinity for Ca²⁺ (K(m) = 12 μM), is dependent on Mg²⁺ or Mn²⁺, and is inhibited by β, γ-imidoadenosine 5'-triphosphate and VO₄^3-, although only slightly (20%) inhibited by high concentrations of mitochondrial inhibitors. Both mechanisms are temperature-dependent, fully reversed by A23187, and higher in the presence of external K⁺. Ca²⁺ loaded in vesicles via ATP-dependent Ca²⁺ uptake is rapidly effluxed upon addition of external Na⁺ (as for Na⁺-Ca²⁺ exchange). Therefore a single population of vesicles exists containing both Ca²⁺ transport mechanisms. The two mechanisms are independent since they accumulate Ca²⁺ additively, are selectively inhibited by monensin and VO₄^3-, and show distinct specificity toward other divalent cations and La³⁺. Although independent, Na⁺ (100 mM) inhibits ATP-dependent Ca²⁺ uptake (K(m) for ATP increased from 40 to 300 μM) in the absence of any net Na⁺ movement. Since Na⁺-Ca²⁺ exchange functions in the synaptosomal plasma membrane, the results suggest that both Ca²⁺ transport mechanisms originate from this membrane and function in the present experiments in inverted plasma membrane vesicles.