Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca(2+) channel related to the animal VGCC/NALCN family

Yeasts and fungi generate Ca(2+) signals in response to environmental stresses through Ca(2+) channels essentially composed of Cch1 and Mid1. Cch1 is homologous to the pore-forming α(1) subunit of animal voltage-gated Ca(2+) channels (VGCCs) and sodium leak channels nonselective (NALCNs), whereas Mid1 is a membrane-associated protein similar to the regulatory α(2)/δ subunit of VGCCs and the regulatory subunit of NALCNs. Although the physiological roles of Cch1/Mid1 channels are known, their molecular regulation remains elusive, including subunit interactions regulating channel functionality. Herein, we identify amino acid residues involved in interactions between the pore-forming Cch1 subunit and the essential regulatory Mid1 subunit of Saccharomyces cerevisiae. In vitro mutagenesis followed by functional assays and co-immunoprecipitation experiments reveal that three residues present in a specific extracellular loop in the repeat III region of Cch1 are required for interaction with Mid1, and that one essential Mid1 residue is required for interaction with Cch1. Importantly, these residues are necessary for Ca(2+) channel activity and are highly conserved in fungal and animal counterparts. We discuss that this unique subunit interaction-based regulatory mechanism for Cch1 differs from that of VGCCs/NALCNs.