Structure and function of the Drosophila protein Big Brain

Abstract

big brain is a neurogenic gene which, when mutated causes defects in cell fate determination during Drosophila neurogenesis through an unknown mechanism. The protein Big Brain (BIB) has sequence identity with the Major Intrinsic Protein family including the water- and ion-conducting Aquaporin channels. We show here that BIB expressed heterologously in Xenopus oocytes is a non-selective monovalent cation channel with permeability to K⁺ > Na⁺ >> TEA⁺. BIB macroscopic conductance, activated in response to endogenous oocyte signaling pathways, was decreased after treatment with 20μM insulin and was enhanced with 10μM lavendustin A, a tyrosine kinase inhibitor. Current activation is not observed in control oocytes or in oocytes expressing a non-functional mutant BIB channel (E71N) that is expressed on the plasma membrane, as confirmed with confocal microscopy and western blotting. Cell-attached patch clamp experiments revealed a novel large conductance (300 ± 30pS) channel in BIB-expressing but not control oocytes. Divalent cations, such as calcium, are important developmental signaling molecules. We found that calcium and barium partially block currents in BIB-expressing oocytes. We further demonstrated that a conserved glutamate (E71) located in transmembrane domain 1 is crucial for channel properties of BIB. Mg²⁺ block was introduced in currents from oocytes expressing the BIB mutant E71D. The carboxy tail of BIB comprises 61% of the channel (431 of 700 residues) and contains sites of potential serine/threonine and tyrosine phosphorylation, SH3 binding domains, PDZ binding domains and three polyglutamine stretches. The importance of the carboxy tail for BIB channel activity was demonstrated by truncation of the channel at two sites. Truncated channels had reduced whole-cell conductance and at least one (Δ317) was not tyrosine phosphorylated. In summary, the results presented in this dissertation provide a novel function of the Drosophila protein Big Brain as a regulated cationic channel, indicate that BIB can participate in tyrosine kinase-regulated transmembrane signaling, and suggest a role for membrane depolarization in the neurogenic function of BIB in early development.