An IP3-activated Ca2+ channel regulates fungal tip growth

Abstract

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-((17β-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.