Summary Neural circuit wiring relies on selective synapse formation whereby a presynaptic release apparatus is matched with its cognate postsynaptic machinery. At metabotropic synapses, the molecular mechanisms underlying this process are poorly understood. In the mammalian retina, rod photoreceptors form selective contacts with rod ON-bipolar cells by aligning the presynaptic voltage-gated Ca2+ channel directing glutamate release (CaV1.4) with postsynaptic mGluR6 receptors. We show this coordination requires an extracellular protein, α2δ4, which complexes with CaV1.4 and the rod synaptogenic mediator, ELFN1, for trans-synaptic alignment with mGluR6. Eliminating α2δ4 in mice abolishes rod synaptogenesis, synaptic transmission to rod ON-bipolar cells, and disrupts postsynaptic mGluR6 clustering. We further find that in rods α2δ4 is crucial for organizing synaptic ribbons and setting CaV1.4 voltage sensitivity. In cones, α2δ4 is essential for CaV1.4 function, but is not required for ribbon organization, synaptogenesis, or synaptic transmission. These findings offer insights into retinal pathologies associated with α2δ4 dysfunction.

Establishment of functional synaptic connections in a selective manner is essential for nervous system operation. In mammalian retinas, rod and cone photoreceptors form selective synaptic connections with different classes of bipolar cells (BCs) to propagate light signals. While there has been progress in elucidating rod wiring, molecular mechanisms used by cones to establish functional synapses with BCs have remained unknown. Using an unbiased proteomic strategy in cone-dominant species, we identified the cell-adhesion molecule ELFN2 to be pivotal for the functional wiring of cones with the ON type of BC. It is selectively expressed in cones and transsynaptically recruits the key neurotransmitter receptor mGluR6 in ON-BCs to enable synaptic transmission. Remarkably, ELFN2 in cone terminals functions in synergy with a related adhesion molecule, ELFN1, and their concerted interplay during development specifies selective wiring and transmission of cone signals. These findings identify a synaptic connectivity mechanism of cones and illustrate how interplay between adhesion molecules and postsynaptic transmitter receptors orchestrates functional synaptic specification in a neural circuit.

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease.

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca channels, respectively, modulate Ca1.2 more potently than Ca1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Ca1.2 into Ca1.3 (Ca1.3+) reduced the IC of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Ca1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Ca1.3+ (Ca1.3+V) further reduced the IC of nifedipine to 42 nM. FPL increased current amplitude with an EC of 854 nM in Ca1.3, 103 nM in Ca1.2, and 99 nM in Ca1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Ca1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 M FPL. FPL had no effect on deactivation of Ca1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC ∼93 M), but did shift the voltage-dependence of activation by ∼-10 mV. We conclude that the M/V variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Ca1.2 and Ca1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined by divergent amino acids in the IIIS5-3P loop.

We investigated the role of Cav1.2 in pancreatic β-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block of KATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Cav current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 μM) stimulated insulin secretion and Ca(2+) transients in INS-1 cells, and Cav1.2/II-III cells were completely blocked by nicardipine (2 μM), but thapsigargin (1 μM) blocked tolbutamide-stimulated secretion and Ca(2+) transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca(2+)] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca(2+) transients in both INS-1 cells and Cav1.2/II-III cells were significantly potentiated by 8-pCPT-2'-O-Me-cAMP (5 μM), FPL-64176 (0.5 μM), or replacement of extracellular Ca(2+) with Sr(2+). Glucose (10 mM) + glucagon-like peptide-1 (10 nM) stimulated discrete spikes in [Ca(2+)]i in the presence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM) stimulated more frequent action potentials in Cav1.2/II-III cells and primary rat β-cells expressing the Cav1.2/II-II loop than in control cells. Further, apamin (1 μM) increased glucose-stimulated action potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channels were not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-III loop of Cav1.2 as a key molecular determinant that couples the channel to Ca(2+)-induced Ca(2+) release and activation of SK channels in pancreatic β-cells.