Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2

Abstract

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.

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

“…, typical of all TMEM16B isoforms (Pifferi et al, 2009aStephan et al, 2009;Ponissery Saidu et al, 2013), but we observed a stationary current similar to the behavior of TMEM16A currents in inside-out patches (Ni et al, 2014;Tien et al, 2014;Yu et al, 2014).…”

“…It is important to note that splice variants have been identified both for TMEM16A (Caputo et al, 2008;Ferrera et al, 2009Ferrera et al, , 2011 and for TMEM16B (Stephan et al, 2009;Ponissery Saidu et al, 2013), and it has been shown that some functional channel properties such as Ca 2+ sensitivity and time and voltage dependence differ among the various isoforms of each protein (Ferrera et al, 2010;Huang et al, 2012;Pedemonte and Galietta, 2014).…”

Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels

“…As previously reported [15,16,32], in TMEM16B wt channel the K 1/2 was slightly voltage-dependent ranging from 4.6 ± 0.2 μM at -50 mV to 2.9 ± 0.1 μM at +50 mV ( n = 7; Wilcoxon signed rank test; Fig 6C). Similar results were obtained for K540Q mutant, whereas R573E and R573E+K540Q showed a significant increase of K 1/2 reaching at -50 mV 28 ± 4 μM and 44 ± 15 μM respectively.…”

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

“…NP_705817.1). This is the retinal isoform with the same start site of the olfactory isoform used in Stephan et al (2009) and contained exon 14 (Ponissery Saidu et al, 2013; named exon 13 in Stephan et al [2009]). 2 µg cDNA was transfected into HEK 293T cells using FuGENE-6 or X-tremeGENE 9 (Roche).…”

“…In TMEM16B, some glutamic acids in the first putative intracellular loop contribute to voltage dependence . A recent study identified splice variants for TMEM16B and found that N-terminal sequences affect Ca 2+ sensitivity (Ponissery Saidu et al, 2013).…”