Cch1 Restores Intracellular Ca2+ in Fungal Cells during Endoplasmic Reticulum Stress

Pathogens endure and proliferate during infection by exquisitely coping with the many stresses imposed by the host to prevent pathogen survival. Recent evidence has shown that fungal pathogens and yeast respond to insults to the endoplasmic reticulum (ER) by initiating Ca2(+) influx across their plasma membrane. Although the high affinity Ca2(+) channel, Cch1, and its subunit Mid1, have been suggested as the protein complex responsible for mediating Ca2(+) influx, a direct demonstration of the gating mechanism of the Cch1 channel remains elusive. In this first mechanistic study of Cch1 channel activity we show that the Cch1 channel from the model human fungal pathogen, Cryptococcus neoformans, is directly activated by the depletion of intracellular Ca2(+) stores. Electrophysiological analysis revealed that agents that enable ER Ca2(+) store depletion promote the development of whole cell inward Ca2(+) currents through Cch1 that are effectively blocked by La3(+) and dependent on the presence of Mid1. Cch1 is permeable to both Ca2(+) and Ba2(+); however, unexpectedly, in contrast to Ca2(+) currents, Ba2(+) currents are steeply voltage-dependent. Cch1 maintains a strong Ca2(+) selectivity even in the presence of high concentrations of monovalent ions. Single channel analysis indicated that Cch1 channel conductance is small, similar to that reported for the Ca2(+) current I-CRAC. This study demonstrates that Cch1 functions as a store-operated Ca2(+)-selective channel that is gated by intracellular Ca2(+) depletion. The inability of cryptococcal cells that lacked the Cch1-Mid1 channel to survive ER stress suggests that Cch1 and its co-regulator, Mid1, are critical players in the restoration of Ca2(+) homeostasis.