Inositol Trisphosphate-dependent and -independent Ca Mobilization Pathways at the Vacuolar Membrane of Candida albicans

doi:10.1074/jbc.270.13.7272

Inositol Trisphosphate-dependent and -independent Ca Mobilization Pathways at the Vacuolar Membrane of Candida albicans(*)

From the Biology Department, University of York, York YO1 5DD, United Kingdom

^§ To whom correspondence should be addressed. Tel.: +44-1904-43-2825; Fax: +44-1904-43-2860.

Abstract

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 A₁. Vacuolar membrane vesicles exhibit two Ca 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 Ca released, the nature of response to successive stimuli, and their respective pharmacological profiles. The InsP₃-gated pathway exhibits a K_0.5 for InsP₃ 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 InsP₃ 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.

Footnotes

↵* This work was supported by a Science and Engineering Research Council-CASE studentship (to C. M. C.) and by Glaxo Group Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

InsP₃

inositol 1,4,5-trisphosphate

Δ

membrane potential

BTP

bis-tris propane

cAMP

cyclic adenosine 3′5′-monophosphate

dantrolene

(1-[(5-[p-nitrophenyl]fur-furylidene)amine]hydantoin)

FCCP

carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone

InsP₂

inositol 4,5-bisphosphate

InsP₄

inositol 1,3,4,5-tetrakisphosphate

Me₂SO

dimethyl sulfoxide

MES

2-[N-morpholino]ethanesulfonic acid

oxonol V

bis-(3-phenyl-5-oxoisooxazol-4-yl)pentamethine oxonol

PIP₂

phosphatidyl inositol 4,5-bisphosphate

Quinacrine

6-chloro-9-{[diethyl-amino)-1-methylbutyl]amino}-2-methoxy-acridine hypochloride

TMB-8

8-(N,N-diethylamino)-octyl-3,4,5-trimethylbenzoate

TPMP

methyltriphenylphosphonium ion (CH₁₈P).
- Received December 7, 1994.