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. 2010 Apr 2;285(14):10951-8.
doi: 10.1074/jbc.M109.056218. Epub 2010 Feb 1.

Cch1 restores intracellular Ca2+ in fungal cells during endoplasmic reticulum stress

Min-Pyo Hong  1 , Kiem Vu, Jennifer Bautos, Angie Gelli
Affiliations

Cch1 restores intracellular Ca2+ in fungal cells during endoplasmic reticulum stress

Min-Pyo Hong et al. J Biol Chem. .

Abstract

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 Ca(2+) influx across their plasma membrane. Although the high affinity Ca(2+) channel, Cch1, and its subunit Mid1, have been suggested as the protein complex responsible for mediating Ca(2+) 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 Ca(2+) stores. Electrophysiological analysis revealed that agents that enable ER Ca(2+) store depletion promote the development of whole cell inward Ca(2+) currents through Cch1 that are effectively blocked by La(3+) and dependent on the presence of Mid1. Cch1 is permeable to both Ca(2+) and Ba(2+); however, unexpectedly, in contrast to Ca(2+) currents, Ba(2+) currents are steeply voltage-dependent. Cch1 maintains a strong Ca(2+) 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 Ca(2+) current I(CRAC). This study demonstrates that Cch1 functions as a store-operated Ca(2+)-selective channel that is gated by intracellular Ca(2+) 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 Ca(2+) homeostasis.

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Figures

FIGURE 1.
FIGURE 1.
Cch1 and Mid1 proteins are expressed in the plasma membrane of HEK293 cells. A, reverse transcription-PCR analysis detected transcripts of CCH1 and MID1 in HEK293 cells previously transfected with CCH1 and MID1 cDNA. Transcripts were not detected in nontransfected (NT) cells. Tubulin is shown as a loading control. B, Mid1 was tagged with a C-terminal GFP tag and visualized by confocal microscopy (center panel). Mid1 is expressed predominantly on the cell surface of HEK293 cells, and Mid1 co-localizes with Cch1 (right panel, merge). Immunofluorescence (Texas Red) of Cch1 (nontagged) revealed a surface distribution in HEK cells (left panel) similar to Mid1. A primary peptide antibody to the C terminus of Cch1 and a Texas Red-conjugated secondary antibody were used to visualize Cch1. Scale bars, 10 μm. C, Western blot analysis of isolated plasma membranes from HEK293 cells expressing Cch1 or Mid1 and revealed polypeptides corresponding to the predicted sizes for Cch1 (∼220 kDa) and Mid1 (monomer, ∼100 kDa; complex, ∼200 kDa). D, Western blot analysis of surface biotinylation of HEK293 cells expressing Cch1 or Mid1 revealed two distinct bands unique to the lane corresponding to Mid1 (monomer, ∼100 kDa; complex, ∼200 kDa). These bands were not detected in nontransfected HEK cells (NT). A protein band corresponding to biotinylated Cch1 could not be detected.
FIGURE 2.
FIGURE 2.
Inward divalent currents through the Cch1 channel are activated by the depletion of Ca2+ stores. A–G, representative traces of time course of whole cell Ca2+ currents through the Cch1 channel. B, trace of Ba2+ currents through Cch1. C and E, Ca2+ and Ba2+ currents measured upon the depletion of Ca2+ stores by the addition of 10 μm BAPTA-AM or with 100 μm thapsigargin (TG) in the patch pipette. D, 10 μm La2+ in the bath solution significantly reducing Ca2+ currents through Cch1. F and G, Ca2+ currents not detected in HEK293 cells expressing either Cch1 alone (F) or Mid1 alone (G). H, average of maximum Ca2+ current levels (−80 mV) measured in HEK293 cells alone (nontransfected, NT) or expressing Cch1, Mid1 or both (n = 5). Data are expressed as means ± S.D. (error bars).
FIGURE 3.
FIGURE 3.
Divalent and monovalent inward currents through the Cch1 channel in HEK293 cells. A, whole cell currents revealed that Cch1 is permeable to both Ca2+ (5 mm) and Ba2+ (5 mm). Arrows indicate Erev (reversal potential) for ECa and EBa. Inward current is carried by Ca2+ or Ba2+, and outward current above +40 mV is carried by Cs+. B, Ba2+ currents are voltage-dependent, whereas Ca2+ currents are voltage-independent. Current levels for Ca2+ and Ba2+ measured at −80 mV and −40 mV were plotted (n = 5). Data are expressed as means ± S.D. (error bars). C, representative single channel record of single channel Ba2+ currents through Cch1. Single channels exhibited long duration openings and a small channel conductance (∼0.08 picosiemens). D, weak Na+ current through the Cch1 channel in the absence of divalent cations. Cch1 maintains a high Ca2+ selectivity in the presence of Ca2+ and high [Na+]. E, Ca2+ or Ba2+ currents were not detected in HEK cells that had been transfected with empty plasmids as shown in the current-voltage (I-V) plot. In addition, the presence of a C-terminal GFP tag on Cch1 did not alter the channel kinetics of Cch1 as shown from the I-V plot measured from HEK cells expressing Cch1 without a GFP tag. E, Ca2+ or Ba2+ channel currents of Cch1 were not affected by the presence of the C-terminal GFP tag on the Cch1 protein as indicated by the I-V plot. Currents were not detected in HEK cells expressing empty plasmids (2) or in nontransfected HEK cells (indicated by flat I-V plots).
FIGURE 4.
FIGURE 4.
Cch1-Mid1 channel is essential for the survival of cryptococcal cells during ER stress. Sensitivity spot assays were used to evaluate the requirement for Cch1 and Mid1 in C. neoformans during drug-induced ER stress conditions. A–D, serially diluted cells (106, 105, 104, 103, 102) from wild type, mid1Δ, cch1Δ, and cch1Δmid1Δ-null mutant strains of C. neoformans were added to YPD agar plates with ER-stressing agents, tunicamycin (75 ng/ml), fluconazole (8 μg/ml), and ketoconazole (180 ng/ml). E–H, ER-stressing agents were added to YPD plates supplemented with BAPTA (cell-impermeant), and spot assays were performed as explained above.
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