Calvert C M, Sanders D
Biology Department, University of York, United Kingdom.
J Biol Chem. 1995 Mar 31;270(13):7272-80. doi: 10.1074/jbc.270.13.7272.
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 H(+)-ATPase, as demonstrated by the sensitivity of Ca2+ uptake to ionophores and the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Vacuolar membrane vesicles exhibit two Ca2+ release pathways: one induced by inositol 1,4,5-trisphosphate (InsP3) 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 InsP3-gated pathway exhibits a K0.5 for InsP3 of 2.4 microM but is not activated by inositol 4,5-bisphosphate or inositol 1,3,4,5-tetrakisphosphate at concentrations up to 50 microM. Ca2+ release by InsP3 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 Ca(2+)-induced Ca2+ release. These findings are discussed in relation to the mechanism of Ca(2+)-mediated cellular signaling in C. albicans and other fungi.
从白色念珠菌原生质体中分离出液泡膜囊泡，并采用标记酶分析法鉴定这些膜来源于液泡。研究了液泡膜上Ca2+摄取和Ca2+释放的机制。液泡膜囊泡对Ca2+的积累可通过H+/Ca2+反向转运产生。内部酸性pH值又由液泡型H(+)-ATP酶产生，这通过Ca2+摄取对离子载体和液泡H(+)-ATP酶抑制剂巴弗洛霉素A1的敏感性得以证明。液泡膜囊泡表现出两种Ca2+释放途径：一种由肌醇1,4,5-三磷酸（InsP3）诱导，另一种由膜内正电压诱导。这两种途径在释放的Ca2+量、对连续刺激的反应性质以及各自的药理学特征方面有所不同。InsP3门控途径对InsP3的K0.5为2.4 microM，但在浓度高达50 microM时，肌醇4,5-二磷酸或肌醇1,3,4,5-四磷酸不会激活该途径。InsP3诱导的Ca2+释放被低分子量肝素部分阻断。电压敏感途径释放Ca2+发生在估计为0至80 mV的生理范围内的膜电位。电压敏感的Ca2+释放途径可被镧系离子和有机通道阻滞剂如钌红和维拉帕米阻断。此外，电压敏感的Ca2+释放途径表现出Ca(2+)-诱导的Ca2+释放。结合白色念珠菌和其他真菌中Ca(2+)介导的细胞信号传导机制对这些发现进行了讨论。
