Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

Abstract

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters-a mechanosensitive channel homolog (MscS), a Ca 2؉ /H ؉ exchange protein (cax), and Ca 2؉ -ATPases (nca-1, nca-2, nca-3)-as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H ؉ -ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca 2؉ levels, indicative of lesions in Ca 2؉ homeostasis. However, the net Ca 2؉ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca 2؉ -selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca 2؉ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca 2؉ ] was elevated. Thus, although Ca 2؉ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654 -661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H ؉ -ATPase activity.

Activation of a TRP-like channel and intracellular calcium dynamics during phospholipase C-mediated cell death

“…Therefore, some caution needs to be taken with regard to the interpretation of results from the inhibitor experiments, especially because the precise targets of these inhibitors in fungal cells have not been characterized in detail. Of the inhibitors employed in our study, several have been previously used in N. crassa – thapsigargin ( Hamam and Lew, 2012 ), bafilomycin A1 ( Bowman et al, 2004 ) and U-73122 ( Silverman-Gavrila and Lew, 2002 ). Ru360, xestospongin C, YM-58483 and ML204 have not been used previously with N. crassa cells but are known to be very selective in animal cells ( Gafni et al, 1997 ; Ishikawa et al, 2003 ; Matlib et al, 1998 ; Miller et al, 2011 ).…”

Calcium homeostasis plays important roles in the internalization and activities of the small synthetic antifungal peptide PAF26

“…We found no significant difference in the resting level of [Ca 2+ [ cyt from the wild type however under unstimulated conditions. Interestingly however, the Δnca‐2 mutant has a membrane potential reversed from the wild type due to a lack of cell surface H + ATPase function (Hamam and Lew, 2012). In S. cerevisiae the H + ATPase is one of the most abundant cell surface proteins (Bagnat et al ., 2001) and makes up a significant amount (up to 10%) of the cell surface in N. crassa (Bowman et al ., 1981).…”

Calcium homeostasis plays important roles in the internalisation and activities of the small synthetic antifungal peptide PAF26

“…We found no significant difference in the resting level of [Ca 2+ [ cyt from the wild 502 type however under unstimulated conditions. Interestingly however, the Δ nca-2 mutant has a 503 membrane potential reversed from the wild type due to a lack of cell surface H + ATPase 504 function (Hamam and Lew 2012). In S. cerevisiae the H + ATPase is one of the most 505 abundant cell surface proteins (Bagnat, Chang et al 2001) and makes up a significant 506 amount (up to 10%) of the cell surface in N. crassa (Bowman, Blasco et al 1981).…”