Vacuolar membrane vesicles were isolated from Candida albicans protoplasts, and marker enzyme assays were employed to identify the membranes as vacuolar in origin. The mechanisms of Ca uptake and Ca release at the vacuolar membrane were investigated. Ca accumulation by vacuolar membrane vesicles can be generated via H/Ca antiport. The inside-acid pH is in turn generated by a vacuolar-type H-ATPase, as demonstrated by the sensitivity of Ca uptake to ionophores and the vacuolar H-ATPase inhibitor bafilomycin A. Vacuolar membrane vesicles exhibit two Ca release pathways: one induced by inositol 1,4,5-trisphosphate (InsP) and the other by inside-positive voltage. These two pathways are distinct with respect to the amount of Ca released, the nature of response to successive stimuli, and their respective pharmacological profiles. The InsP-gated pathway exhibits a K for InsP of 2.4 µM but is not activated by inositol 4,5-bisphosphate or inositol 1,3,4,5-tetrakisphosphate at concentrations up to 50 µM. Ca release by InsP is blocked partially by low molecular weight heparin. Ca released by the voltage-sensitive pathway occurs at membrane potentials estimated to be over a physiological range from 0 to 80 mV. The voltage-sensitive Ca release pathway can be blocked by lanthanide ions and organic channel blockers such as ruthenium red and verapamil. Furthermore, the voltage-sensitive Ca release pathway exhibits Ca-induced Ca release. These findings are discussed in relation to the mechanism of Ca-mediated cellular signaling in C. albicans and other fungi.

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