INOSITOL TRISPHOSPHATE-DEPENDENT AND TRISPHOSPHATE-INDEPENDENT CA2+ MOBILIZATION PATHWAYS AT THE VACUOLAR MEMBRANE OF CANDIDA-ALBICANS

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 Ca2+ uptake and Ca2+ release at the vacuolar membrane were investigated, Ca2+ accumulation by vacuolar membrane vesicles can be generated via H+/Ca2+ antiport. The inside-acid pH is in turn generated by a vacuolar-type HC-ATPase, as demonstrated by the sensitivity of Ca2+ uptake to ionophores and the vacuolar H+-ATPase inhibitor bafilomycin A(1). Vacuolar membrane vesicles exhibit two Ca2+ release pathways: one induced by inositol 1,4,5-trisphosphate (InsP(3)) and the other by inside-positive voltage. These two pathways are distinct with respect to the amount of Ca2+ released, the nature of response to successive stimuli, and their respective pharmacological profiles, The InsP(3)-gated pathway exhibits a K-0.5 for InsP(3) of 2.4 mu M but is not activated by inositol 4,5-bisphosphate or inositol 1,3,4,5-tetrakisphosphate at concentrations up to 50 mu M. Ca2+ release by InsP(3) is blocked partially by low molecular weight heparin, Ca2+ 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 Ca2+ release pathway can be blocked by lanthanide ions and organic channel blockers such as ruthenium red and verapamil. Furthermore, the voltage-sensitive Ca2+ release pathway exhibits Ca2+-induced Ca2+ release. These findings are discussed in relation to the mechanism of Ca2+-mediated cellular signaling in C. albicans and other fungi.