A visually guided swim assay for mouse models of human retinal disease recapitulates the multi-luminance mobility test in humans.

PURPOSE: The purpose of this study was to develop a visually guided swim assay (VGSA) for measuring vision in mouse retinal disease models comparable to the multi-luminance mobility test (MLMT) utilized in human clinical trials. METHODS: Three mouse retinal disease models were studied: Bardet-Biedl syndrome type 1 (Bbs1M390R/M390R), n = 5; Bardet-Biedl syndrome type 10 (Bbs10-/-), n = 11; and X linked retinoschisis (retinoschisin knockout; Rs1-KO), n = 5. Controls were normally-sighted mice, n = 10. Eyeless Pax6Sey-Dey mice, n = 4, were used to determine the performance of animals without vision in VGSA. RESULTS: Eyeless Pax6Sey-Dey mice had a VGSA time-to-platform (TTP) 7X longer than normally-sighted controls (P < 0.0001). Controls demonstrated no difference in their TTP in both lighting conditions; the same was true for Pax6Sey-Dey. At 4-6 M, Rs1-KO and Bbs10-/- had longer TTP in the dark than controls (P = 0.0156 and P = 1.23 × 10-8, respectively). At 9-11 M, both BBS models had longer TTP than controls in light and dark with times similar to Pax6Sey-Dey (P < 0.0001), demonstrating progressive vision loss in BBS models, but not in controls nor in Rs1-KO. At 1 M, Bbs10-/- ERG light-adapted (cone) amplitudes were nonrecordable, resulting in a floor effect. VGSA did not reach a floor until 9-11 M. ERG combined rod/cone b-wave amplitudes were nonrecordable in all three mutant groups at 9-11 M, but VGSA still showed differences in visual function. ERG values correlate non-linearly with VGSA, and VGSA measured the continual decline of vision. CONCLUSION: ERG is no longer a useful endpoint once the nonrecordable level is reached. VGSA differentiates between different levels of vision, different ages, and different disease models even after ERG is nonrecordable, similar to the MLMT in humans.

Photoreceptor Ion Channels in Signaling and Disease.

Photoreceptors (PRs) in the neural retina convert photon capture into an electrical signal that is communicated across a chemical synapse to second-order neurons in the retina and on through the rest of the visual pathway. This information is decoded in the visual cortex to create images. The activity of PRs depends on the concerted action of several voltage-gated ion channels that will be discussed in this chapter.

Mouse all-cone retina models of Cav1.4 synaptopathy.

The voltage-gated calcium channel, Cav1.4 is localized to photoreceptor ribbon synapses and functions both in molecular organization of the synapse and in regulating release of synaptic vesicles. Mutations in Cav1.4 subunits typically present as either incomplete congenital stationary night blindness or a progressive cone-rod dystrophy in humans. We developed a cone-rich mammalian model system to further study how different Cav1.4 mutations affect cones. RPE65 R91W KI; Nrl KO "Conefull" mice were crossed to Cav1.4 α1F or α2δ4 KO mice to generate the "Conefull:α1F KO" and "Conefull:α2δ4 KO" lines. Animals were assessed using a visually guided water maze, electroretinogram (ERG), optical coherence tomography (OCT), and histology. Mice of both sexes and up to six-months of age were used. Conefull: α1F KO mice could not navigate the visually guided water maze, had no b-wave in the ERG, and the developing all-cone outer nuclear layer reorganized into rosettes at the time of eye opening with degeneration progressing to 30% loss by 2-months of age. In comparison, the Conefull: α2δ4 KO mice successfully navigated the visually guided water maze, had a reduced amplitude b-wave ERG, and the development of the all-cone outer nuclear layer appeared normal although progressive degeneration with 10% loss by 2-months of age was observed. In summary, new disease models for studying congenital synaptic diseases due to loss of Cav1.4 function have been created.

AAV2/4-RS1 gene therapy in the retinoschisin knockout mouse model of X-linked retinoschisis.

OBJECTIVE: To evaluate efficacy of a novel adeno-associated virus (AAV) vector, AAV2/4-RS1, for retinal rescue in the retinoschisin knockout (Rs1-KO) mouse model of X-linked retinoschisis (XLRS). Brinzolamide (Azopt®), a carbonic anhydrase inhibitor, was tested for its ability to potentiate the effects of AAV2/4-RS1. METHODS: AAV2/4-RS1 with a cytomegalovirus (CMV) promoter (2x1012 viral genomes/mL) was delivered to Rs1-KO mice via intravitreal (N = 5; 1µL) or subretinal (N = 21; 2µL) injections at postnatal day 60-90. Eleven mice treated with subretinal therapy also received topical Azopt® twice a day. Serial full field electroretinography (ERG) was performed starting at day 50-60 post-injection. Mice were evaluated using a visually guided swim assay (VGSA) in light and dark conditions. The experimental groups were compared to untreated Rs1-KO (N = 11), wild-type (N = 12), and Rs1-KO mice receiving only Azopt® (N = 5). Immunofluorescence staining was performed to assess RS1 protein expression following treatment. RESULTS: The ERG b/a ratio was significantly higher in the subretinal plus Azopt® (p<0.0001), subretinal without Azopt® (p = 0.0002), and intravitreal (p = 0.01) treated eyes compared to untreated eyes. There was a highly significant subretinal treatment effect on ERG amplitudes collectively at 7-9 months post-injection (p = 0.0003). Cones showed more effect than rods. The subretinal group showed improved time to platform in the dark VGSA compared to untreated mice (p<0.0001). RS1 protein expression was detected in the outer retina in subretinal treated mice and in the inner retina in intravitreal treated mice. CONCLUSIONS: AAV2/4-RS1 shows promise for improving retinal phenotype in the Rs1-KO mouse model. Subretinal delivery was superior to intravitreal. Topical brinzolamide did not improve efficacy. AAV2/4-RS1 may be considered as a potential treatment for XLRS patients.

Pentameric assembly of the Kv2.1 tetramerization domain.

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristics of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily lacking a representative structure of the T1 domain is the Kv2 family. Here, X-ray crystallography was used to solve the structure of the human Kv2.1 T1 domain. The structure is similar to those of other T1 domains, but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer, as determined by inline SEC-MALS-SAXS and negative-stain electron microscopy. The Kv2.1 T1-T1 interface involves electrostatic interactions, including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. It is shown that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains, which may reflect the versatility of Kv2.1, which can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

Identification of HCN1 as a 14-3-3 client.

Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed, as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding peptides is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of mouse S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1.

Cone-Driven Retinal Responses Are Shaped by Rod But Not Cone HCN1.

Signal integration of converging neural circuits is poorly understood. One example is in the retina where the integration of rod and cone signaling is responsible for the large dynamic range of vision. The relative contribution of rods versus cones is dictated by a complex function involving background light intensity and stimulus temporal frequency. One understudied mechanism involved in coordinating rod and cone signaling onto the shared retinal circuit is the hyperpolarization activated current (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels expressed in rods and cones. Ih opposes membrane hyperpolarization driven by activation of the phototransduction cascade and modulates the strength and kinetics of the photoreceptor voltage response. We examined conditional knock-out (KO) of HCN1 from mouse rods using electroretinography (ERG). In the absence of HCN1, rod responses are prolonged in dim light which altered the response to slow modulation of light intensity both at the level of retinal signaling and behavior. Under brighter intensities, cone-driven signaling was suppressed. To our surprise, conditional KO of HCN1 from mouse cones had no effect on cone-mediated signaling. We propose that Ih is dispensable in cones because of the high level of temporal control of cone phototransduction. Thus, HCN1 is required for cone-driven retinal signaling only indirectly by modulating the voltage response of rods to limit their output.SIGNIFICANCE STATEMENT Hyperpolarization gated hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry a feedback current that helps to reset light-activated photoreceptors. Using conditional HCN1 knock-out (KO) mice we show that ablating HCN1 from rods allows rods to signal in bright light when they are normally shut down. Instead of enhancing vision this results in suppressing cone signaling. Conversely, ablating HCN1 from cones was of no consequence. This work provides novel insights into the integration of rod and cone signaling in the retina and challenges our assumptions about the role of HCN1 in cones.

Differential impact of Kv8.2 loss on rod and cone signaling and degeneration.

Heteromeric Kv2.1/Kv8.2 channels are voltage-gated potassium channels localized to the photoreceptor inner segment. They carry IKx, which is largely responsible for setting the photoreceptor resting membrane potential. Mutations in Kv8.2 result in childhood-onset cone dystrophy with supernormal rod response (CDSRR). We generated a Kv8.2 knockout (KO) mouse and examined retinal signaling and photoreceptor degeneration to gain deeper insight into the complex phenotypes of this disease. Using electroretinograms, we show that there were delayed or reduced signaling from rods depending on the intensity of the light stimulus, consistent with reduced capacity for light-evoked changes in membrane potential. The delayed response was not seen ex vivo where extracellular potassium levels were controlled by the perfusion buffer, so we propose the in vivo alteration is influenced by genotype-associated ionic imbalance. We observed mild retinal degeneration. Signaling from cones was reduced but there was no loss of cone density. Loss of Kv8.2 altered responses to flickering light with responses attenuated at high frequencies and altered in shape at low frequencies. The Kv8.2 KO line on an all-cone retina background had reduced cone-driven ERG b wave amplitudes and underwent degeneration. Altogether, we provide insight into how a deficit in the dark current affects the health and function of photoreceptors.

A Comparison of the Primary Sensory Neurons Used in Olfaction and Vision.

Vision, hearing, smell, taste, and touch are the tools used to perceive and navigate the world. They enable us to obtain essential resources such as food and highly desired resources such as mates. Thanks to the investments in biomedical research the molecular unpinning's of human sensation are rivaled only by our knowledge of sensation in the laboratory mouse. Humans rely heavily on vision whereas mice use smell as their dominant sense. Both modalities have many features in common, starting with signal detection by highly specialized primary sensory neurons-rod and cone photoreceptors (PR) for vision, and olfactory sensory neurons (OSN) for the smell. In this chapter, we provide an overview of how these two types of primary sensory neurons operate while highlighting the similarities and distinctions.

A dual role for Cav1.4 Ca2+ channels in the molecular and structural organization of the rod photoreceptor synapse.

Synapses are fundamental information processing units that rely on voltage-gated Ca2+ (Cav) channels to trigger Ca2+-dependent neurotransmitter release. Cav channels also play Ca2+-independent roles in other biological contexts, but whether they do so in axon terminals is unknown. Here, we addressed this unknown with respect to the requirement for Cav1.4 L-type channels for the formation of rod photoreceptor synapses in the retina. Using a mouse strain expressing a non-conducting mutant form of Cav1.4, we report that the Cav1.4 protein, but not its Ca2+ conductance, is required for the molecular assembly of rod synapses; however, Cav1.4 Ca2+ signals are needed for the appropriate recruitment of postsynaptic partners. Our results support a model in which presynaptic Cav channels serve both as organizers of synaptic building blocks and as sources of Ca2+ ions in building the first synapse of the visual pathway and perhaps more broadly in the nervous system.

Antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent.

Benzalkonium chloride (BC) is a quaternary ammonium antimicrobial agent used in a variety of applications. In this work, BC was prepared into deep eutectic solvent (DES) with acrylic acid (AA) or methacrylic acid (MA). Within the newly prepared DES, BC is responsible for antimicrobial properties, while AA and MA are responsible for polymerization. Three types of microorganisms, E. coli (gram-negative bacilli), S. aureus (gram-positive cocci) and C. albicans (fungi), were assessed for antimicrobial properties through agar diffusion test. DES viscosity measurements and polymerizations were also conducted to assist the antimicrobial performance analysis. From this study, stronger antimicrobial effectiveness of BC-AA DES towards S. aureus and C. albicans was observed, while smaller inhibition zone widths were obtained for BC-AA DES polymer compared to BC-AA DES monomer which may due to the limited active component transportation after polymerization. When changing AA to MA, increased structural complexity and decreased linearity may limit the molecule movement thus reduce the inhibition zone width, which could be proved by the calculated activation energy results. Accurately determined eutectic ratio of DES is recommended to get optimized drug release control. This work offers a new sight for preparation of antimicrobial materials with stronger effectiveness and limited release.

Rescue of Rod Synapses by Induction of Cav Alpha 1F in the Mature Cav1.4 Knock-Out Mouse Retina.

Purpose: Cav1.4 is a voltage-gated calcium channel clustered at the presynaptic active zones of photoreceptors. Cav1.4 functions in communication by mediating the Ca2+ influx that triggers neurotransmitter release. It also aids in development since rod ribbon synapses do not form in Cav1.4 knock-out mice. Here we used a rescue strategy to investigate the ability of Cav1.4 to trigger synaptogenesis in both immature and mature mouse rods. Methods: In vivo electroporation was used to transiently express Cav α1F or tamoxifen-inducible Cav α1F in a subset of Cav1.4 knock-out mouse rods. Synaptogenesis was assayed using morphologic markers and a vision-guided water maze. Results: We found that introduction of Cav α1F to knock-out terminals rescued synaptic development as indicated by PSD-95 expression and elongated ribbons. When expression of Cav α1F was induced in mature animals, we again found restoration of PSD-95 and elongated ribbons. However, the induced expression of Cav α1F led to diffuse distribution of Cav α1F in the terminal instead of being clustered beneath the ribbon. Approximately a quarter of treated animals passed the water maze test, suggesting the rescue of retinal signaling in these mice. Conclusions: These data confirm that Cav α1F expression is necessary for rod synaptic terminal development and demonstrate that rescue is robust even in adult animals with late stages of synaptic disease. The degree of rod synaptic plasticity seen here should be sufficient to support future vision-restoring treatments such as gene or cell replacement that will require photoreceptor synaptic rewiring.

α2δ-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses.

α2δ-4 is an auxiliary subunit of voltage-gated Cav1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the CACNA2D4 gene encoding α2δ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of α2δ-4, we used genome editing to generate an α2δ-4 knock-out (α2δ-4 KO) mouse. In male and female α2δ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in α2δ-4 KO retina. In reconstructions of α2δ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in α2δ-4 KO mice correlates with a progressive loss of Cav1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in α2δ-4 KO mice and better under photopic than scotopic conditions. We conclude that α2δ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with CACNA2D4 mutations.SIGNIFICANCE STATEMENT In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for α2δ-4, a subunit of voltage-gated Ca2+ channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca2+ channels are progressively lost and that rod and cone synapses are disrupted in mice that lack α2δ-4. Our results suggest that alterations in presynaptic Ca2+ signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the CACNA2D4 gene encoding α2δ-4.

Analysis of 14-3-3 isoforms expressed in photoreceptors.

The 14-3-3 family of proteins has undergone considerable expansion in higher eukaryotes with humans and mice expressing seven isoforms (ß, ε, η, γ, θ, ζ, and σ) from seven distinct genes (YWHAB, YWAHE, YWHAH, YWHAG, YWHAQ, YWHAZ, and SFN). Growing evidence indicates that while highly conserved, these isoforms are not entirely functionally redundant as they exhibit unique tissue expression profiles, subcellular localization, and biochemical functions. A key limitation in our understanding of 14-3-3 biology lies in our limited knowledge of cell-type specific 14-3-3 expression. Here we provide a characterization of 14-3-3 expression in whole retina and isolated rod photoreceptors using reverse-transcriptase digital droplet PCR. We find that all 14-3-3 genes with the exception of SFN are expressed in mouse retina with YWHAQ and YWHAE being the most highly expressed. Rod photoreceptors are enriched in YWHAE (14-3-3 ε). Immunohistochemistry revealed that 14-3-3 ε and 14-3-3 ζ exhibit unique distributions in photoreceptors with 14-3-3 ε restricted to the inner segment and 14-3-3 ζ localized to the outer segment. Our data demonstrates that, in the retina, 14-3-3 isoforms likely serve specific functions as they exhibit unique expression levels and cell-type specificity. As such, future investigations into 14-3-3 function in rod photoreceptors should be centered on 14-3-3 ε and 14-3-3 ζ, depending on the subcellular region of question.

Bacterial Cellulose Ionogels as Chemosensory Supports.

To fully leverage the advantages of ionic liquids for many applications, it is necessary to immobilize or encapsulate the fluids within an inert, robust, quasi-solid-state format that does not disrupt their many desirable, inherent features. The formation of ionogels represents a promising approach; however, many earlier approaches suffer from solvent/matrix incompatibility, optical opacity, embrittlement, matrix-limited thermal stability, and/or inadequate ionic liquid loading. We offer a solution to these limitations by demonstrating a straightforward and effective strategy toward flexible and durable ionogels comprising bacterial cellulose supports hosting in excess of 99% ionic liquid by total weight. Termed bacterial cellulose ionogels (BCIGs), these gels are prepared using a facile solvent-exchange process equally amenable to water-miscible and water-immiscible ionic liquids. A suite of characterization tools were used to study the preliminary (thermo)physical and structural properties of BCIGs, including no-deuterium nuclear magnetic resonance, differential scanning calorimetry, thermogravimetric analysis, scanning electron microscopy, and X-ray diffraction. Our analyses reveal that the weblike structure and high crystallinity of the host bacterial cellulose microfibrils are retained within the BCIG. Notably, not only can BCIGs be tailored in terms of shape, thickness, and choice of ionic liquid, they can also be designed to host virtually any desired active, functional species, including fluorescent probes, nanoparticles (e.g., quantum dots, carbon nanotubes), and gas-capture reagents. In this paper, we also present results for fluorescent designer BCIG chemosensor films responsive to ammonia or hydrogen sulfide vapors on the basis of incorporating selective fluorogenic probes within the ionogels. Additionally, a thermometric BCIG hosting the excimer-forming fluorophore 1,3-bis(1-pyrenyl)propane was devised which exhibited a ratiometric (two-color) fluorescence output that responded precisely to changes in local temperature. The ionogel approach introduced here is simple and has broad generality, offering intriguing potential in (bio)analytical sensing, catalysis, membrane separations, electrochemistry, energy storage devices, and flexible electronics and displays.

Incorporation of antibacterial agent derived deep eutectic solvent into an active dental composite.

OBJECTIVE: To incorporate an antibacterial agent derived deep eutectic solvent (DES) into a dental resin composite, and investigate the resulting mechanical properties and antibacterial effects. METHOD: The DES was derived from benzalkonium chloride (BC) and acrylic acid (AA) and was incorporated into the dental resin composite through rapid mixing. A three-point bending test was employed to measure the flexural strength of the composite. An agar diffusion test was used to investigate antibacterial activity. Artificial (accelerated) aging was undertaken by immersing the composites in buffer solutions at an elevated temperature for up to 4 weeks. UV-vis spectrophotometry and NMR analysis were conducted to study BC release from the composite. Finally, the biocompatibility of the composite materials was evaluated using osteoblast cell culture for 7 days. Results were compared to those of a control composite which contained no BC. RESULT: The DES-incorporated composite (DES-C) displayed higher flexural strength than a similar BC-incorporated composite BC (BC-C) for the same level of BC. The inclusion of BC conferred antibacterial activity to both BC-containing composites, although BC-C produced larger inhibition halos than DES-C at the same loading of BC. Control composites which contained no BC showed negligible antibacterial activity. After artificial aging, the DES-C composite showed better maintenance of the mechanical properties of the control compared with BC-C, although a decrease was observed during the three-point bending test, particularly upon storage at elevated temperatures. No BC release was detected in the aged solutions of DES-C, whereas the BC-C showed a linear increase in BC release with storage time. Significantly, cell viability results indicated that DES-C has better biocompatibility than BC-C. SIGNIFICANCE: The incorporation of a BC-based DES into a dental resin composite provides a new strategy to develop antibacterial dental materials with better biocompatibility and longer effective lifetimes without sacrificing the intrinsic mechanical properties of the composite structure.

Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses.

PURPOSE: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. METHODS: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. RESULTS: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.

Mechanism for Selective Synaptic Wiring of Rod Photoreceptors into the Retinal Circuitry and Its Role in Vision.

In the retina, rod and cone photoreceptors form distinct connections with different classes of downstream bipolar cells. However, the molecular mechanisms responsible for their selective connectivity are unknown. Here we identify a cell-adhesion protein, ELFN1, to be essential for the formation of synapses between rods and rod ON-bipolar cells in the primary rod pathway. ELFN1 is expressed selectively in rods where it is targeted to the axonal terminals by the synaptic release machinery. At the synapse, ELFN1 binds in trans to mGluR6, the postsynaptic receptor on rod ON-bipolar cells. Elimination of ELFN1 in mice prevents the formation of synaptic contacts involving rods, but not cones, allowing a dissection of the contributions of primary and secondary rod pathways to retinal circuit function and vision. We conclude that ELFN1 is necessary for the selective wiring of rods into the primary rod pathway and is required for high sensitivity of vision.

Identification of a VxP Targeting Signal in the Flagellar Na+ /K+ -ATPase.

Na(+) /K(+) -ATPase (NKA) participates in setting electrochemical gradients, cardiotonic steroid signaling and cellular adhesion. Distinct isoforms of NKA are found in different tissues and subcellular localization patterns. For example, NKA α1 is widely expressed, NKA α3 is enriched in neurons and NKA α4 is a testes-specific isoform found in sperm flagella. In some tissues, ankyrin, a key component of the membrane cytoskeleton, can regulate the trafficking of NKA. In the retina, NKA and ankyrin-B are expressed in multiple cell types and immunostaining for each is striking in the synaptic layers. Labeling for NKA is also prominent along the inner segment plasma membrane (ISPM) of photoreceptors. NKA co-immunoprecipitates with ankyrin-B, but on a subcellular level colocalization of these two proteins varies dependent on the cell type. We used transgenic Xenopus laevis tadpoles to evaluate the subcellular trafficking of NKA in photoreceptors. GFP-NKA α3 and α1 are localized to the ISPM, but α4 is localized to outer segments (OSs). We identified a VxP motif responsible for the OS targeting by using a series of chimeric and mutant NKA constructs. This motif is similar to previously identified ciliary targeting motifs. Given the structural similarities between OSs and flagella, our findings shed light on the subcellular targeting of this testes-specific NKA isoform.

An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors.

PURPOSE: Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. METHODS: We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. RESULTS: We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. CONCLUSIONS: We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane.