title: Hyperactive and hypoactive mutations in Cch1, a yeast homologue of the voltage-gated calcium-channel pore-forming subunit.
abstract: The yeast Saccharomyces cerevisiae CCH1 gene encodes a homologue of the pore-forming α1 subunit of mammalian voltage-gated calcium channels. Cch1 cooperates with Mid1, a candidate for a putative, functional homologue of the mammalian regulatory subunit α2/δ, and is essential for Ca(2+) influx induced by several stimuli. Here, we characterized two mutant alleles of CCH1, CCH1* (or CCH1-star, carrying four point mutations: V49A, N1066D, Y1145H and N1330S) and cch1-2 (formerly designated mid3-2). The product of CCH1* displayed a marked increase in Ca(2+) uptake activity in the presence and absence of α-factor, and its increased activity was still dependent on Mid1. Mutations in CCH1* did not affect its susceptibility to regulation by calcineurin. In addition, not only was the N1066D mutation in the cytoplasmic loop between domains II and III responsible for the increased activity of Cch1*, but also substitution of another negatively charged amino acid Glu for Asn(1066) resulted in a significant increase in the Ca(2+) uptake activity of Cch1. This is the first report of a hyperactive mutation in Cch1. On the other hand, the cch1-2 allele possesses the P1228L mutation located in the extracellular S1-S2 linker of domain III. The Pro(1228) residue is highly conserved from fungi to humans, and the P1228L mutation led to a partial loss in Cch1 function, but did not affect the localization and expression of Cch1. The results extend our understanding of the structure-function relationship and functional regulation of Cch1.
authors.affiliation: Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Teng
authors.affiliation: Biomembrane Laboratory, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Iida
authors.affiliation: Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Imai
authors.affiliation: Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Nakano
authors.affiliation: Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tada
authors.affiliation: Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Iida
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: C108765:CCH1 protein, S cerevisiae; D015220:Calcium Channels; C090463:MID1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; D002118:Calcium