Cytosolic calcium homeostasis in fungi: roles of plasma membrane transport and intracellular sequestration of calcium.
Cytosolic free calcium ([Ca2+]c) has been measured in the mycelial fungus Neurospora crassa with Ca2(+)-selective microelectrodes. The mean value of [Ca2+]c is 92 +/- 15 nM and it is insensitive to external pH values between 5.8 and 8.4. Simultaneous measurement of membrane potential enables the electrochemical potential difference for Ca2+ across the plasma membrane to be estimated as about -60 kJ.mol-1-a value that cannot be sustained either by a simple Ca2(+)-ATPase, or, in alkaline conditions, by straightforward H+/Ca2+ exchange with a stoichiometric ratio of less than 5 H+/Ca2+. We propose that the most likely alternative mechanism of Ca2+ efflux is ATP-driven H+/Ca2+. In accord with this proposal, depletion of the ATP level from 2.5 to 0.5 mM by CN- elicits an increase in [Ca2+]c, but only in alkaline conditions in which the putative H+/Ca2(+)-ATPase would be selectively stalled. The insensitivity of Ca2+ homeostasis to CN- in more acid conditions implies that the Km (ATP) of the transport system is 100 microM or less. The increase in [Ca2+]c in the presence of CN- at pH 8.4 (50 nM.min-1) is compared with 45Ca2+ influx (0.62 mM.min-1) under the same conditions. The proportion of entering Ca2+ remaining free in the cytosol is only 8 x 10(-5), and since the concentration of available chelation sites on Ca2(+)-binding proteins is unlikely to exceed 100 microM, a major role for the fungal vacuole in short-term Ca2+ homeostasis is indicated. This notion is supported by the observation that cytosolic Ca2+ homeostasis is disrupted by a protonophore, which rapidly abolishes the driving force (a transmembrane pH difference) for Ca2+ uptake into fungal vacuoles.
Miller
A J
AJ
Biology Department, University of York, United Kingdom.
Vogg
G
G
Sanders
D
D
D000255:Adenosine Triphosphate / Q000378:metabolism; D017920:Antiporters; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008839:Microelectrodes; D009492:Neurospora crassa / Q000378:metabolism*; D013816:Thermodynamics
D017920:Antiporters; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; C027614:calcium-hydrogen antiporters; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium