Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²âº channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.

Anoctamin 2/TMEM16B: a calcium-activated chloride channel in olfactory transduction.

In vertebrate olfactory transduction, a Ca(2+)-dependent Cl(-) efflux greatly amplifies the odorant response. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca(2+) into the cilia. The Ca(2+) activates a Cl(-) current that, in the presence of a maintained elevated intracellular Cl(-) concentration, produces an efflux of Cl(-) ions and amplifies the depolarization. In this review, we summarize evidence supporting the hypothesis that anoctamin 2/TMEM16B is the main, or perhaps the only, constituent of the Ca(2+)-activated Cl(-) channels involved in olfactory transduction. Indeed, studies from several laboratories have shown that anoctamin 2/TMEM16B is expressed in the ciliary layer of the olfactory epithelium, that there are remarkable functional similarities between currents in olfactory sensory neurons and in HEK 293 cells transfected with anoctamin 2/TMEM16B, and that knockout mice for anoctamin 2/TMEM16B did not show any detectable Ca(2+)-activated Cl(-) current. Finally, we discuss the involvement of Ca(2+)-activated Cl(-) channels in the transduction process of vomeronasal sensory neurons and the physiological role of these channels in olfaction.

Pannexin 1 Is Critically Involved in Feedback from Horizontal Cells to Cones.

Retinal horizontal cells (HCs) feed back negatively to cone photoreceptors and in that way generate the center/surround organization of bipolar cell receptive fields. The mechanism by which HCs inhibit photoreceptors is a matter of debate. General consensus exists that horizontal cell activity leads to the modulation of the cone Ca-current. This modulation has two components, one fast and the other slow. Several mechanisms for this modulation have been proposed: a fast ephaptic mechanism, and a slow pH mediated mechanism. Here we test the hypothesis that the slow negative feedback signal from HCs to cones is mediated by Panx1 channels expressed at the tips of the dendrites of horizontal cell. We generated zebrafish lacking Panx1 and found that the slow component of the feedback signal was strongly reduced in the mutants showing that Panx1 channels are a fundamental part of the negative feedback pathway from HCs to cones.

Assessment of the olfactory function in Italian patients with type 3 von Willebrand disease caused by a homozygous 253 Kb deletion involving VWF and TMEM16B/ANO2.

Type 3 Von Willebrand disease is an autosomal recessive disease caused by the virtual absence of the von Willebrand factor (VWF). A rare 253 kb gene deletion on chromosome 12, identified only in Italian and German families, involves both the VWF gene and the N-terminus of the neighbouring TMEM16B/ANO2 gene, a member of the family named transmembrane 16 (TMEM16) or anoctamin (ANO). TMEM16B is a calcium-activated chloride channel expressed in the olfactory epithelium. As a patient homozygous for the 253 kb deletion has been reported to have an olfactory impairment possibly related to the partial deletion of TMEM16B, we assessed the olfactory function in other patients using the University of Pennsylvania Smell Identification Test (UPSIT). The average UPSIT score of 4 homozygous patients was significantly lower than that of 5 healthy subjects with similar sex, age and education. However, 4 other members of the same family, 3 heterozygous for the deletion and 1 wild type, had a slightly reduced olfactory function indicating that socio-cultural or other factors were likely to be responsible for the observed difference. These results show that the ability to identify odorants of the homozygous patients for the deletion was not significantly different from that of the other members of the family, showing that the 253 kb deletion does not affect the olfactory performance. As other genes may compensate for the lack of TMEM16B, we identified some predicted functional partners from in silico studies of the protein-protein network of TMEM16B. Calculation of diversity for the corresponding genes for individuals of the 1000 Genomes Project showed that TMEM16B has the highest level of diversity among all genes of the network, indicating that TMEM16B may not be under purifying selection and suggesting that other genes in the network could compensate for its function for olfactory ability.

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel.

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca(2+)-activated Cl(-) channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl(-) with other anions (PX/PCl) was SCN(-) > I(-) > NO3 (-) > Br(-) > Cl(-) > F(-) > gluconate. When external Cl(-) was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca(2+) were modified according to the sequence of permeability ratios, with anions more permeant than Cl(-) slowing both activation and deactivation and anions less permeant than Cl(-) accelerating them. Moreover, replacement of external Cl(-) with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl(-) with SCN(-) shifted G-V to more negative potentials. Dose-response relationships for Ca(2+) in the presence of different extracellular anions indicated that the apparent affinity for Ca(2+) at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca(2+) in the presence of intracellular SCN(-) also increased compared with that in Cl(-). Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop.

Ca(2+)-activated Cl(-) channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure-function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca(2+) dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca(2+) concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates (386)EEEEE(390) and (399)EYE(401). The EYE deletion did not significantly modify the apparent Ca(2+) dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca(2+) affinity but modified the voltage dependence, shifting the conductance-voltage relations toward more positive voltages. These findings indicate that glutamates E367 and (386)EEEEE(390) in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure-function study for this channel.