Calretinin Regulates Ca2+-dependent Inactivation and Facilitation of Cav2.1 Ca2+ Channels through a Direct Interaction with the α12.1 Subunit

Voltage-gated Ca(v)2.1 Ca2+ channels undergo dual modulation by Ca2+, Ca2+-dependent inactivation (CDI), and Ca2+-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 Ca2+-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to alpha(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 alpha(1)2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca2+-dependent. alpha(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 alpha(1)2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of alpha(1)2.1 with that from the Ca(v)1.2 alpha(1) subunit (alpha(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 alpha(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 Ca2+ signaling and neuronal excitability.