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 Ca ²⁺-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.