The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the β-lactam producerPenicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treatedAspergillus nidulanshyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface ofA. nidulansprotoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism inA. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity ofA. nidulansstrain FGSC1053, which carries a dominant-interfering mutation infadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Ca_v1.2) of the L-type Ca²⁺ channel. Glutathionylation of Ca_v1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Ca_v1.2 is responsible for altered function. We directly assessed the function of purified human Ca_v1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Ca_v1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses.