TMEM16B, a Novel Protein With Calcium-Dependent Chloride Channel Activity, Associates With a Presynaptic Protein Complex in Photoreceptors

Abstract

Purpose: : Calcium-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. The TMEM16 family of transmembrane proteins has recently been associated with calcium-dependent chloride channel function. The aim of this study is to clone TMEM16B and to analyze its role in photoreceptor physiology.

Methods: : Molecular genetics and biocomputational methods were used to isolate TMEM16B cDNAs. Expression analysis was performed by RT-PCR. Antibodies against mouse TMEM16B were generated and used for Western blotting and immunohistochemistry in wild-type and Mpp4^-/- mice. Binding assays were applied to determine interaction with synaptic proteins. Halide-sensitive YFP assays and whole-cell patch clamp recordings were performed to study calcium-dependent chloride channel activity.

Results: : We cloned human and mouse TMEM16B and show that it is abundantly expressed in the retina. TMEM16B co-localizes with adaptor proteins PSD95, VELI3 and MPP4 at mouse photoreceptor synaptic membranes and is capable to interact with PDZ domains of PSD95. Immunohistochemistry reveals that TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. Finally, we demonstrate that TMEM16B possesses calcium-dependent chloride channel activity when expressed in mammalian cells.

Keywords: photoreceptors • synapse • ion channels