title: Activity of the calcium channel pore Cch1 is dependent on a modulatory region of the subunit Mid1 in Cryptococcus neoformans.
abstract: Calcium (Ca(2+))-mediated signaling events in fungal pathogens such as Cryptococcus neoformans are central to physiological processes, including those that mediate stress responses and promote virulence. The Cch1-Mid1 channel (CMC) represents the only high-affinity Ca(2+) channel in the plasma membrane of fungal cells; consequently, cryptococci cannot survive in low-Ca(2+) environments in the absence of CMC. Previous electrophysiological characterization revealed that Cch1, the predicted channel pore, and Mid1, a binding partner of Cch1, function as a store-operated Ca(2+)-selective channel gated by depletion of endoplasmic reticulum (ER) Ca(2+) stores. Cryptococci lacking CMC did not survive ER stress, indicating its critical role in restoring Ca(2+) homeostasis. Despite the requirement for Mid1 in promoting Ca(2+) influx via Cch1, identification of the role of Mid1 remains elusive. Here we show that the C-terminal tail of Mid1 is a modulatory region that impinges on Cch1 channel activity directly and mediates the trafficking of Mid1 to the plasma membrane. This region consists of the last 24 residues of Mid1, and the functional expression of Mid1 in a human embryonic cell line (HEK293) and in C. neoformans is dependent on this domain. Substitutions of arginine (R619A) or cysteine (C621A) in the modulatory region failed to target Mid1 to the plasma membrane and prevented CMC activity. Interestingly, loss of a predicted protein kinase C (PKC)-phosphorylated serine residue (S605A) had no effect on Mid1 trafficking but did alter the kinetics of Cch1 channel activity. Thus, establishment of Ca(2+) homeostasis in C. neoformans is dependent on a modulatory domain of Mid1.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, Davis, Davis, California, USA.
authors.forename: Min-Pyo
authors.initials: MP
authors.lastname: Hong
authors.forename: Kiem
authors.initials: K
authors.lastname: Vu
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Bautos
authors.forename: Rick
authors.initials: R
authors.lastname: Tham
authors.forename: Mantana
authors.initials: M
authors.lastname: Jamklang
authors.forename: John P
authors.initials: JP
authors.lastname: Uhrig
authors.forename: Angie
authors.initials: A
authors.lastname: Gelli
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling; D003455:Cryptococcus neoformans / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid
chemical_list: D015220:Calcium Channels; D005656:Fungal Proteins; D021122:Protein Subunits