Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.
SUMMARY Studies of the zebrafish epithalamus have provided recent insights into the development of left-right brain asymmetry, which is crucial to normal human brain function. The habenular nuclei (Hb) of zebrafish are robustly asymmetric, with dense elaboration of neuropil only in the left lateral subnucleus. Because this feature is tightly correlated with asymmetric expression of K+ channel tetramerization domain-containing proteins 12.1 and 12.2 (Kctd12.1/12.2), we screened for Kctd12.1-interacting proteins to identify molecular mechanisms leading to neuropil asymmetry, and uncovered a novel interaction between Kctd12.1 and Unc-51-like kinase 2 (Ulk2). We show here that knockdown of Ulk2 or overexpression of Kctd12 proteins reduce asymmetric neuropil elaboration. Conversely, overexpression of Ulk2 or mutation of kctd12 genes cause excess neuropil elaboration. We conclude that Ulk2 activity promotes neuropil elaboration while Kctd12 proteins limit Ulk2 activity asymmetrically. This work describes a regulatory mechanism for neuronal process extension that may be conserved in other developmental contexts in addition to the epithalamus.
Animals detect odorous chemicals through specialized olfactory sensory neurons (OSNs) that transduce odorants into neural electrical signals. We identified a novel and evolutionarily conserved protein, cilia-and flagella-associated protein 69 (CFAP69), in mice that regulates olfactory transduction kinetics. In the olfactory epithelium, CFAP69 is enriched in OSN cilia, where olfactory transduction occurs. Bioinformatic analysis suggests that a large portion of CFAP69 can form Armadillo-type ␣-helical repeats, which may mediate protein-protein interactions. OSNs lacking CFAP69, remarkably, displayed faster kinetics in both the on and off phases of electrophysiological responses at both the neuronal ensemble level as observed by electroolfactogram and the single-cell level as observed by single-cell suction pipette recordings. In single-cell analysis, OSNs lacking CFAP69 showed faster response integration and were able to fire APs more faithfully to repeated odor stimuli. Furthermore, both male and female mutant mice that specifically lack CFAP69 in OSNs exhibited attenuated performance in a buried food pellet test when a background of the same odor to the food pellet was present even though they should have better temporal resolution of coding olfactory stimulation at the peripheral. Therefore, the role of CFAP69 in the olfactory system seems to be to allow the olfactory transduction machinery to work at a precisely regulated range of response kinetics for robust olfactory behavior.
Figure 1. Characterization of Ano2 mRNA variants present in mouse olfactory epithelium. (A) Diagram summarizing the 5 Ano2 exon splicing structure. The green sections indicate the most 5 AUG translation start codons, and the subsequent black bars indicate predicted protein-coding sequence. The variants containing exons 1a and 2 are less abundant in the olfactory epithelium than the variants containing the newly discovered exon 1b (red), as determined in B and C. The five forward (F1-F5) and one reverse (R1) PCR primer-binding sites are indicated as arrows. Given two alternative 5 ends and alternative splicing of exon 4, these mRNA variants may encode up to four ANO2 isoforms, named on the right. (B and C) Ethidium bromide-stained agarose gels showing RT-PCR products with primers specific for each 5 variant of Ano2. Primer F1 is universal for both variants, F2 is specific for exon 1b, F3 and F4 are specific for exon 2, and F5 is specific for exon 1a. The reverse primer (R1) was the same for all PCR reactions. The expected PCR product sizes are 267, 409, 350, 511, and 830 bp, respectively. Quantification of the bands by densitometry is shown in Fig. S1 C.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.