The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.

Mutations in the bestrophin-1 (Best1) gene are linked to several kinds of macular degeneration in both humans and dogs. Although bestrophins have been shown clearly to be Cl− ion channels, it is controversial whether Cl− channel dysfunction can explain the diseases. It has been suggested that bestrophins are multi-functional proteins: they may regulate voltage-gated Ca2+ channels in addition to functioning as Cl− channels. Here we show that hBest1 differentially modulates Cav1.3 (L-type) voltage-gated Ca2+ channels through association with the Cavβ subunit. In transfected HEK-293 cells, hBest1 inhibited Cav1.3. Inhibition of Cav1.3 was not observed in the absence of the β subunit. Also, the hBest1 C-terminus binds to Cavβ subunits, suggesting that the effect of hBest1 was mediated by the Cavβ subunit. The region of hBest1 responsible for the effect was localized to a region (amino acids 330 − 370) in the cytoplasmic C-terminus that contains a predicted SH3-binding domain that is not present in other bestrophin subtypes. Mutation of Pro330 and Pro334 abolished the effects of hBest1 on Cav1.3. The effect was specific to hBest1: it was not observed with mBest1, -2, or -3. Wild type hBest1 and the disease-causing mutants R92S, G299R, and D312N inhibited Cav currents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates Cav channels by interacting with the Cavβ subunit and altering channel availability. Our findings reveal a novel function of bestrophin in regulation of Cav channels and suggest a possible mechanism for the role of hBest1 in macular degeneration.