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.
Odorants cause Ca(2+) to rise in olfactory sensory neurons (OSNs) first within the ciliary compartment, then in the dendritic knob, and finally in the cell body. Ca(2+) not only excites but also produces negative feedback on the transduction pathway. To relieve this Ca(2+)-dependent adaptation, Ca(2+) must be cleared from the cilia and dendritic knob by mechanisms that are not well understood. This work focuses on the roles of plasma membrane calcium pumps (PMCAs) through the use of inhibitors and mice missing 1 of the 4 PMCA isoforms (PMCA2). We demonstrate a significant contribution of PMCAs in addition to contributions of the Na(+)/Ca(2+) exchanger and endoplasmic reticulum (ER) calcium pump to the rate of calcium clearance after OSN stimulation. PMCAs in neurons can shape the Ca(2+) signal. We discuss the contributions of the specific PMCA isoforms to the shape of the Ca(2+) transient that controls signaling and adaptation in OSNs.
The systematic exploration of a series of triazole-based agonists of the cation channel insect odorant receptor is reported. The structure-activity relationships of independent sections of the molecules are examined. Very small changes to the compound structure were found to exert a large impact on compound activity. Optimal substitutions were combined using a “mix-and-match” strategy to produce best-in-class compounds that are capable of potently agonizing odorant receptor activity and may form the basis for the identification of a new mode of insect behavior modification.
Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao 1 , originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca 2+ -activated Cl -channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body [2][3][4] .In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors 5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors 6 . The suction pipette technique was later adapted to also record from ORNs 7,8 . Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body 9 . The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.
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.
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