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doi: 10.1242/10.1242/jcs.00180

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An IP₃-activated Ca²⁺ channel regulates fungal tip growth

Biology Department, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada

^* Author for correspondence (e-mail: planters{at}yorku.ca)

Accepted 23 September 2002

Hyphal extension in fungi requires a tip-high Ca²⁺ gradient, which is generated and maintained internally by inositol (1,4,5)-trisphosphate (IP₃)-induced Ca²⁺ release from tip-localized vesicles and subapical Ca²⁺ sequestration. Using the planar bilayer method we demonstrated the presence of two types of IP₃-activated Ca²⁺ channels in Neurospora crassa membranes with different conductances: one low (13 picosiemens), the other high (77 picosiemens). On sucrose density gradients the low conductance channel co-localized with endoplasmic reticulum and plasma membrane, and the high conductance channel co-localized with vacuolar membranes. We correlated the effect of inhibitors on channel activity with their effect on hyphal growth and Ca²⁺ gradients. The inhibitor of IP₃-induced Ca²⁺ release, 2-aminoethoxidiphenylborate (2-APB), inhibits both channels, while heparin, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8) and dantrolene inhibit only the large conductance channel. Because 2-APB inhibits hyphal growth and dissipates the tip-high cytosolic [Ca²⁺] gradient, whereas heparin microinjection, TMB-8 and dantrolene treatments do not affect growth, we suggest that the small conductance channel generates the obligatory tip-high Ca²⁺ gradient during hyphal growth. Since IP₃ production must be catalyzed by tip-localized phospholipase C, we show that a number of phospholipase C inhibitors [neomycin, 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]- 1H-pyrrole-2,5-dione (U-73122) (but not the inactive pyrrolidine U-73343), 3-nitrocoumarin] inhibit hyphal growth and affect, similarly to 2-APB, the location of vesicular Ca²⁺ imaged by chlortetracycline staining.

Key words: Fungal tip growth, IP₃ receptor, Ca²⁺ gradient, Phospholipase C, Ca²⁺ channels, Planar lipid membrane

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