Abstract P080: Bestrophin: A Potential Mediator of Cardiac Calcium Handling

Bestrophins are a family of transmembrane proteins expressed in heart. We reported that Bestrophin 1 and 3 cloned from mouse heart function as calcium-activated chloride channels. We hypothesized that bestrophin channels may function in a macromolecule complex similar to other cardiac ion channels. To identify proteins that bind Bestrophin 3 channel, we screened a mouse heart cDNA library using 4 different cytosolic cDNA fragments as bait. Yeast two-hybrid assays were performed with the GAL4 system. Using bait consisting of C-terminus amino acid residues 386 to 669 of the Best3 channel (Best3-C2), we obtained positive interactions with the histidine-rich calcium binding protein (hrc). Hrc is a component of the SR complex involved in calcium handling in the heart. Eleven independent clones represented a fragment encoding the cysteine rich domain of hrc previously shown to interact with triadin. To further map the interaction sites between Best3-C2 and hrc, we subdivided this region into three fragments and tested these baits for interaction with hrc. Two Best3 fragments (residues 541–600 and residues 601–669) displayed strong interaction with hrc, while Best3 bait (residues 386–540) failed to interact. This region of Best3 contains a KEKE protein binding motif. In a similar manner C-terminal Best 1 fragments were confirmed to also interact with hrc. In this study we report that Bestrophin channels interact with hrc, a cardiac calcium handling protein. Calcium-activated chloride channels play a critical role in excitation-contraction coupling in the SR by balancing charge movement during calcium release and reuptake. Recent evidence confirms Bestrophin 1 is located in the ER membrane in association with Stim1 and facilitates calcium cycling. Future in vivo investigations will examine the role of bestrophins as potential mediators of calcium handling in heart.