Vacuolar-type proton ATPase is required for maintenance of apicobasal polarity of embryonic visceral endoderm

The endocytic compartments keep their interior acidic through the inward flow of protons and anions from the cytosol. Acidification is mediated by a proton pump known as vacuolar-type ATPase (V-ATPase) and transporters conferring anion conductance to the organellar membrane. In this study, we analysed the phenotype of mouse embryos lacking the V-ATPase c-subunit. The mutant embryos differentiated embryonic epithelial tissues, primitive endoderm, epiblast, and extraembryonic ectoderm; however, the organisation of these epithelia was severely affected. The apical-basal polarity in the visceral endoderm layer was not properly established in the mutant embryos, resulting in abnormal epithelial morphology. Thus, the function of V-ATPase is imperative for the establishment and/or maintenance of epithelial cell polarity, which is required for early embryogenesis.

The Crystal Structure of Bromide-Bound GtACR1 Reveals a Pre-Activated State in the Transmembrane Anion Tunnel

The crystal structure of the light-gated anion channel GtACR1 reported in our previous research article (Li et al., 2019) revealed a continuous tunnel traversing the protein from extracellular to intracellular pores. We proposed the tunnel as the conductance channel closed by three constrictions: C1 in the extracellular half, mid-membrane C2 containing the photoactive site, and C3 on the cytoplasmic side. Reported here, the crystal structure of bromide-bound GtACR1 reveals structural changes that relax the C1 and C3 constrictions, including a novel salt-bridge switch mechanism involving C1 and the photoactive site. These findings indicate that substrate binding induces a transition from an inactivated state to a pre-activated state in the dark that facilitates channel opening by reducing free energy in the tunnel constrictions. The results provide direct evidence that the tunnel is the closed form of the channel of GtACR1 and shed light on the light-gated channel activation mechanism.Impact StatementSubstrate-induced structural changes in GtACR1 provide new insight into the chemical mechanism of natural light-gated anion conductance, and facilitate its optimization for photoinhibition of neuron firing in optogenetics.

SLC26A7 constitutes the thiocyanate-selective anion conductance of the basolateral membrane of the retinal pigment epithelium

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl− between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN−), a property that does not correspond with any of the Cl− channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN− permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl− channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN− conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN−-selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.

Membrane insertion of chromogranin B for granule maturation in regulated secretion

ABSTRACTRegulated secretion serves responses to specific stimuli in eukaryotes. An anion conductance was found essential for maturation and acidification of secretory granules four decades ago, but its genetic identity was unknown. We now demonstrate that chromogranin B (CHGB), an obligate granule protein, constitutes the long-sought anion channel. High-pressure freezing immuno-electron microscopy and biochemical assays showed native CHGB in close proximity to secretory granule membranes, and its membrane-bound and soluble forms both reconstituted Cl- channels. Release of secretory granules delivered CHGB clusters to plasma membranes, which dominate whole-cell anion conductance. Intragranular pH measurements and cargo maturation assays found that CHGB channels supported proinsulin - insulin conversion and dopamine-loading in neuroendocrine cells. β-cells from Chgb-/- mice exhibited significant granule deacidification, accounting for hyperproinsulinemia, altered glucose-tolerance response and lower dopamine concentration in chromaffin granules in these animals. Membrane insertion of well-conserved CHGB is thus indispensable for granule maturation in exocrine, endocrine and neuronal cells.HighlightsNative CHGB is amphipathic and distributes in the lumen and membranes of secretory granules with contrastingly different destinies and functions.Native CHGB, once delivered to cell surface via granule exocytosis, dominates anion conductance in plasma membranes.CHGB channels facilitate granule acidification and cargo maturation in cultured and primary neuroendocrine cells.CHGB channels from bovine, rat and mouse cells all serve the long-missing, intra-organellar anion shunt pathway in the secretory granules for regulated secretion.

Crystal structure of a natural light-gated anion channelrhodopsin

The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

Mouse retinal pigment epithelial cells exhibit a thiocyanate-selective conductance

The basolateral membrane anion conductance of the retinal pigment epithelium (RPE) is a key component of the transepithelial Cl− transport pathway. Although multiple Cl− channels have been found to be expressed in the RPE, the components of the resting Cl− conductance have not been identified. In this study, we used the patch-clamp method to characterize the ion selectivity of the anion conductance in isolated mouse RPE cells and in excised patches of RPE basolateral and apical membranes. Relative permeabilities ( PA/ PCl) calculated from reversal potentials measured in intact cells under bi-ionic conditions were as follows: SCN− >> ClO4− > [Formula: see text] > I− > Br− > Cl− >> gluconate. Relative conductances ( GA/ GCl) followed a similar trend of SCN− >> ClO4− > [Formula: see text] > I− > Br− ≈Cl− >> gluconate. Whole cell currents were highly time-dependent in 10 mM external SCN−, reflecting collapse of the electrochemical potential gradient due to SCN− accumulation or depletion intracellularly. When the membrane potential was held at −120 mV to minimize SCN− accumulation in cells exposed to 10 mM SCN−, the instantaneous current reversed at −90 mV, revealing that PSCN/ PCl is approximately 500. Macroscopic current recordings from outside-out patches demonstrated that both the basolateral and apical membranes exhibit SCN− conductances, with the basolateral membrane having a larger SCN− current density and higher relative permeability for SCN−. Our results suggest that the RPE basolateral and apical membranes contain previously unappreciated anion channels or electrogenic transporters that may mediate the transmembrane fluxes of SCN− and Cl−.

Crystal Structure of a Natural Light-Gated Anion Channelrhodopsin

ABSTRACTThe anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

Novel organelle anion channels formed by chromogranin B drive normal granule maturation in endocrine cells

All endocrine cells need an anion conductance for maturation of secretory granules. Identity of this family of anion channels has been elusive for forty years. We now show that a family of granule proteins, CHGB, serves the long-sought conductance. CHGB interacts with membranes through two amphipathic helices, and forms a chloride channel with a large conductance and high anion selectivity. Fast kinetics and high cooperativity suggest that CHGB tetramerizes to form a functional channel. Nonconducting mutants separate CHGB channel function in granule maturation from its role in granule biogenesis. In neuroendocrine cells, CHGB channel and a H+-ATPase drive normal insulin maturation inside or catecholamine loading into secretory granules. Tight membrane-association of CHGB after exocytotic release of secretory granules separates its intracellular functions from the extracellular functions accomplished by its proteolytic peptides. CHGB-null mice show impairment of granule acidification in pancreatic beta-cells due to lack of anion conductance. These findings together support that the phylogenetically conserved CHGB proteins constitute a fifth family of chloride channels that function in various endocrine cells.