Abstract:

<p>Mutations of human , encoding a Ca-activated Cl channel (hBest1), cause macular degenerative disorders. Best1 homolog structures reveal an evolutionarily conserved channel architecture highlighted by two landmark restrictions (named the &quot;neck&quot; and &quot;aperture&quot;, respectively) in the ion conducting pathway, suggesting a unique dual-switch gating mechanism, which, however, has not been characterized well. Using patch clamp and crystallography, we demonstrate that both the neck and aperture in hBest1 are Ca-dependent gates essential for preventing channel leakage resulting from Ca-independent, spontaneous gate opening. Importantly, three patient-derived mutations (D203A, I205T and Y236C) lead to Ca-independent leakage and elevated Ca-dependent anion currents due to enhanced opening of the gates. Moreover, we identify a network of residues critically involved in gate operation. Together, our results suggest an indispensable role of the neck and aperture of hBest1 for channel gating, and uncover disease-causing mechanisms of hBest1 gain-of-function mutations.</p>