Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca 2ϩ -dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca 2ϩ -dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca 2ϩ -dependent chloride channel in the photoreceptor synapse.

Changes in cell function occur by specific patterns of intracellular Ca2+, activating Ca2+-sensitive proteins. The anoctamin (TMEM16) protein family has Ca2+-dependent ion channel activity, which provides transmembrane ion transport, and/or Ca2+-dependent phosphatidyl-scramblase activity. Using amino acid sequence analysis combined with measurements of ion channel function, we clarified the so far unknown Ano4 function as Ca2+-dependent, non-selective monovalent cation channel; heterologous Ano4 expression in HEK293 cells elicits Ca2+ activated conductance with weak selectivity of K+ > Na+ > Li+. Endogenously expressed Ca2+-dependent cation channels in the retinal pigment epithelium were identified as Ano4 by KO mouse-derived primary RPE cells and siRNA against Ano4. Exchanging a negatively charged amino acid in the putative pore region (AA702–855) into a positive one (E775K) turns Ano4-elicited currents into Cl− currents evidencing its importance for ion selectivity. The molecular identification of Ano4 as a Ca2+-activated cation channel advances the understanding of its role in Ca2+ signaling.