Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels.

Anoctamin-1 (ANO1) (TMEM16A) is a calcium-activated chloride channel that plays critical roles in diverse physiological processes, such as sensory transduction and epithelial secretion. ANO1 levels have been shown to be altered under physiological and pathological conditions, although the molecular mechanisms that control ANO1 protein levels remain unclear. The ubiquitin-proteasome system is known to regulate the levels of numerous ion channels, but little information is available regarding whether and how ubiquitination regulates levels of ANO1. Here, we showed that two E3 ligases, TRIM23 and TRIM21, physically interact with the C terminus of ANO1. In vitro and in vivo assays demonstrated that whereas TRIM23 ubiquitinated ANO1 leading to its stabilization, TRIM21 ubiquitinated ANO1 and induced its degradation. Notably, ANO1 regulation by TRIM23 and TRIM21 is involved in chemical-induced pain sensation, salivary secretion, and heart-rate control in mice, and TRIM23 also mediates ANO1 upregulation induced by epidermal growth factor treatment. Our results suggest that these two antagonistic E3 ligases act together to control ANO1 expression and function. Our findings reveal a previously unrecognized mechanism for regulating ANO1 protein levels and identify a potential molecular link between ANO1 regulation, epidermal growth factor, and other signaling pathways.

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

The complex of TRIP-Br1 and XIAP ubiquitinates and degrades multiple adenylyl cyclase isoforms.

Adenylyl cyclases (ACs) generate cAMP, a second messenger of utmost importance that regulates a vast array of biological processes in all kingdoms of life. However, almost nothing is known about how AC activity is regulated through protein degradation mediated by ubiquitination or other mechanisms. Here, we show that transcriptional regulator interacting with the PHD-bromodomain 1 (TRIP-Br1, Sertad1), a newly identified protein with poorly characterized functions, acts as an adaptor that bridges the interaction of multiple AC isoforms with X-linked inhibitor of apoptosis protein (XIAP), a RING-domain E3 ubiquitin ligase. XIAP ubiquitinates a highly conserved Lys residue in AC isoforms and thereby accelerates the endocytosis and degradation of multiple AC isoforms in human cell lines and mice. XIAP/TRIP-Br1-mediated degradation of ACs forms part of a negative-feedback loop that controls the homeostasis of cAMP signaling in mice. Our findings reveal a previously unrecognized mechanism for degrading multiple AC isoforms and modulating the homeostasis of cAMP signaling.

Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial ligand-gated anion channel, are associated with the lethal genetic disease cystic fibrosis. The CFTR G551D mutation impairs ATP hydrolysis and thereby makes CFTR refractory to cAMP stimulation. Both wild-type (WT) and G551D CFTR have been implicated in regulatory volume decrease (RVD), but the underlying mechanism remains incompletely understood. Here, we show that the channel activity of both WT and G551D CFTR is directly stimulated by mechanical perturbation induced by cell swelling at the single-channel, cellular, and tissue levels. Hypotonicity activated CFTR single channels in cell-attached membrane patches and WT-CFTR-mediated short-circuit current (Isc) in Calu-3 cells, and this was independent of Ca(2+)and cAMP/PKA signaling. Genetic suppression and ablation but not G551D mutation of CFTR suppressed the hypotonicity- and stretch-inducedIscin Calu-3 cells and mouse duodena. Moreover, ablation but not G551D mutation of the CFTR gene inhibited the RVD of crypts isolated from mouse intestine; more importantly, CFTR-specific blockers markedly suppressed RVD in both WT- and G551D CFTR mice, demonstrating for the first time that the channel activity of both WT and G551D CFTR is required for epithelial RVD. Our findings uncover a previously unrecognized mechanism underlying CFTR involvement in epithelial RVD and suggest that the mechanosensitivity of G551D CFTR might underlie the mild phenotypes resulting from this mutation.-Xie, C., Cao, X., Chen, X, Wang, D., Zhang, W. K., Sun, Y., Hu, W., Zhou, Z., Wang, Y., Huang, P. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.