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Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

ABSTRACT: The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

SUBMITTER: Yurtsever Z

PROVIDER: S-EPMC3516759 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

Yurtsever Zeynep Z Sala-Rabanal Monica M Randolph David T DT Scheaffer Suzanne M SM Roswit William T WT Alevy Yael G YG Patel Anand C AC Heier Richard F RF Romero Arthur G AG Nichols Colin G CG Holtzman Michael J MJ Brett Tom J TJ

The Journal of biological chemistry 20121030 50

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefi ...[more]

PMID: 23112050

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Project description:The transmembrane 16 (TMEM16) family contains 10 subtypes, and the function of each protein is different. TMEM16A is a calcium-activated chloride channel involved in physiological and pathological situations. Liquiritigenin is an aglycone derived from Glycyrrhiza glabra, and it is generated via the metabolism of enterobacterial flora. It has been known that liquiritigenin reduces pain sensation involving TMEM16A activation in primary sensory neurons. In addition, other pharmacological effects of liquiritigenin in physiological functions involving TMEM16A have been reported. However, the relationship between TMEM16A and liquiritigenin is still unknown. Therefore, we hypothesized that TMEM16A is inhibited by liquiritigenin. To confirm this hypothesis, we investigated the effect of liquiritigenin on TMEM16A currents evoked by intracellular free calcium in HEK293T cells transfected with TMEM16A. In this study, we found that liquiritigenin inhibited the mouse and human TMEM16A currents. To further confirm its selectivity, we also investigated its pharmacological effects on other ion channels, including transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), which are non-selective cation channels involved in pain sensation. However, liquiritigenin did not inhibit the currents of TRPV1 and TRPA1 induced by capsaicin and allyl isothiocyanate, respectively. Therefore, our findings indicate that selective TMEM16A inhibition could be one molecular mechanism that explains liquiritigenin-induced pain reduction. Additionally, we also investigated the inhibitory effects of estrogens on TMEM16A because liquiritigenin reportedly binds to the estrogen receptor. In this study, a pregnancy-dependent estrogen, estriol, significantly inhibited TMEM16A. However, the efficacy was weak. Although there is a possibility that TMEM16A activity could be suppressed during pregnancy, the physiological significance seems to be small. Thus, the inhibitory effect of estrogen might not be significant under physiological conditions. Furthermore, we investigated the effect of dihydrodaidzein, which is an analog of liquiritigenin that has a hydroxyphenyl at different carbon atom of pyranose. Dihydrodaidzein also inhibited mouse and human TMEM16A. However, the inhibitory effects were weaker than those of liquiritigenin. This suggests that the efficacy of TMEM16A antagonists depends on the hydroxyl group positions. Our finding of liquiritigenin-dependent TMEM16A inhibition could connect the current fragmented knowledge of the physiological and pathological mechanisms involving TMEM16A and liquiritigenin.

Dataset Information

Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

Publications

Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

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