TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals

Abstract

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.

Ca2+-activated Cl− currents are dispensable for olfaction

“…Consistent with previous data [21][22][23] , western blots indicated that Ano2 is most highly expressed in olfactory epithelium (MOE and VNO) and eye tissue (Fig. 1a,b).…”

“…Our N-terminal antibody did not detect a truncated protein in Ano2 -/-tissue, suggesting the occurrence of nonsensemediated mRNA decay, instability of the truncated protein, or both. Ano2 -/-mice were born at Mendelian ratio; they grew, mated, and survived normally and lacked an immediately apparent phenotype.Consistent with previous data [21][22][23] , western blots indicated that Ano2 is most highly expressed in olfactory epithelium (MOE and VNO) and eye tissue (Fig. 1a,b).…”

“…5d,f), but not at 13 µM free Ca 2+ (Fig. 5g,i) similar to data reported for Ca 2+ -activated Cl -currents for heterologously expressed Ano1 and Ano2 [20][21][22]33,34 and for frog OSNs 7 . We could not detect these Cl -currents in Ano2 -/-OSNs (Fig.…”

Activation of Ca2+‐activated Cl− channel ANO1 by localized Ca2+ signals

“…2011) and photoreceptors (Stohr et al . 2009), apparently has a different Ca 2+ source preference as compared to ANO1. Huang and colleagues (Huang et al .…”

“…2009; Stohr et al . 2009) subunits identified as bona fide CaCCs (see Pedemonte & Galietta, 2014 for review). For example, ANO1 (TMEM16A), reconstituted in an expression system, reproduced key features of native CaCC currents such as higher permeability to I − over Cl − , micromolar Ca 2+ sensitivity, outwardly rectifying voltage dependence and sensitivity to the CaCC blockers 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB), niflumic acid (NFA) and 4,4′‐diisothio‐cyanostilbene‐2,2′‐disulfonic acid (DIDS) (Hartzell et al .…”