Osmolarity was adjusted with sucrose to 305 mOsm for all solutions. the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312323) reduced the apparent Ca2+affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350390) that is required for Ca2+activation and plays a role ICOS in current rundown. These experiments identify a region of hBest1 (312323) that is involved in the gating of hBest1 by Ca2+and suggest a model in which Ca2+binding to EF1 activates the channel in a process that requires the acidic domain (293308) and another regulatory domain (350390). Many of the 100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+. == INTRODUCTION == Mutations in human bestrophin-1 (hBest1) have been shown to be responsible for several retinopathies including Best vitelliform macular dystrophy (Petrukhin et al., 1998;Marquardt et al., 1998), adult-onset macular dystrophy (Seddon et al., 2001), autosomal dominant vitreochoidopathy (Yardley et al., 2004), autosomal recessive bestrophinopathy (Burgess et al., 2008), and canine multifocal retinopathy (Guziewicz et al., 2007). hBest1 is a Clion channel that is activated by intracellular Ca2+with a Kdof 150 GS-626510 nM (for review seeHartzell et al., 2008). The structures and mechanisms responsible for Ca2+sensitivity, however, remain unknown. It seems likely GS-626510 that activation of the bestrophin channel is mediated by Ca2+binding directly to the channel or to an accessory Ca2+-binding subunit, such as calmodulin (CaM), because channels are activated in excised patches containing hBest4 or dBest1 in the absence of ATP (Chien et al., 2006;Tsunenari et al., 2006). GS-626510 In this article, we focus on the potential role of a conserved region in the cytoplasmic C terminus immediately after the last predicted transmembrane domain in regulation of hBest1 by Ca2+. The most common and well-understood kind of Ca2+-binding sites in proteins are EF hand motifs (for review seeNelson and Chazin, 1998;Lewit-Bentley and Rety, 2000; andGifford et al., 2007). These are subdivided into the canonical EF hand as found in the CaM family and the pseudoEF hand structures as found in the S100-like family. The Ca2+-binding loop of the canonical EF hand is formed primarily by side chain oxygens, whereas the pseudoEF hand Ca2+-binding site is formed primarily by backbone carbonyl oxygens. In addition, there are other Ca2+-binding motifs, like the C2 domain, which have a less well-defined primary structure (Brose et al., 1995). The fact that Ca2+can be coordinated by backbone carbonyl oxygens as well as by side chain oxygens means that at the present time prediction of Ca2+-binding sites from primary sequence data is challenging (Zhou et al., 2006). In any case, there are no obvious GS-626510 canonical EF hand motifs detected in hBest1 by motif-searching algorithms such as Pfam 22 (http://pfam.sanger.ac.uk/). In trying to fathom the Ca2+-binding site of bestrophins, it has been noted that bestrophins exhibit a highly conserved region immediately after the last predicted transmembrane domain that contains a high concentration of acidic amino acids that might be involved in Ca2+binding (Fig. 1) (Tsunenari et al., 2006;Hartzell et al., 2008). This region exhibits some similarity to the Ca2+bowl of the.