Block by Extracellular Divalent Cations of Drosophila Big Brain Channels Expressed in Xenopus Oocytes

Abstract

Drosophila Big Brain (BIB) is a transmembrane protein encoded by the neurogenic gene big brain ( bib), which is important for early development of the fly nervous system. BIB expressed in Xenopus oocytes is a monovalent cation channel modulated by tyrosine kinase signaling. Results here demonstrate that the BIB conductance shows voltage- and dose-dependent block by extracellular divalent cations Ca 2+ and Ba 2+ but not by Mg 2+ in wild-type channels. Site-directed mutagenesis of negatively charged glutamate (Glu 274) and aspartate (Asp 253) residues had no effect on divalent cation block. However, mutation of a conserved glutamate at position 71 (Glu 71) in the first transmembrane domain (M1) altered channel properties. Mutation of Glu 71 to Asp introduced a new sensitivity to block by extracellular Mg 2+; substitutions with asparagine or glutamine decreased whole-cell conductance; and substitution with lysine compromised plasma membrane expression. Block by divalent cations is important in other ion channels for voltage-dependent function, enhanced signal resolution, and feedback regulation. Our data show that the wild-type BIB conductance is attenuated by external Ca 2+, suggesting that endogenous divalent cation block might be relevant for enhancing signal resolution or voltage dependence for the native signaling process in neuronal cell fate determination.