title: Calretinin regulates Ca2+-dependent inactivation and facilitation of Ca(v)2.1 Ca2+ channels through a direct interaction with the α12.1 subunit.
abstract: Voltage-gated Ca(v)2.1 Ca(2+) channels undergo dual modulation by Ca(2+), Ca(2+)-dependent inactivation (CDI), and Ca(2+)-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca(2+)-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α(1)2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α(1)2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca(2+)-dependent. α(1)2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca(v)2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca(v)2.1 CDI and increased CDF. CR binding to α(1)2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α(1)2.1 with that from the Ca(v)1.2 α(1) subunit (α(1)1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α(1)2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca(v)2.1, which may have major consequences for Ca(2+) signaling and neuronal excitability.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Christel
authors.forename: Raphael
authors.initials: R
authors.lastname: Schaer
authors.forename: Shiyi
authors.initials: S
authors.lastname: Wang
authors.forename: Thomas
authors.initials: T
authors.lastname: Henzi
authors.forename: Lisa
authors.initials: L
authors.lastname: Kreiner
authors.forename: Detlev
authors.initials: D
authors.lastname: Grabs
authors.forename: Beat
authors.initials: B
authors.lastname: Schwaller
authors.forename: Amy
authors.initials: A
authors.lastname: Lee
mesh_terms: D000818:Animals; D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*
chemical_list: D064032:Calbindin 2; D002118:Calcium; D020864:Calcium Channels, N-Type; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; C576764:CALB2 protein, human; C576511:Calb2 protein, mouse