Dataset Information

Identification and characterization of two ankyrin-B isoforms in mammalian heart.

ABSTRACT:

Aims

Excitation-contraction coupling in cardiomyocytes requires the proper targeting and retention of membrane proteins to unique domains by adaptor proteins like ankyrin-B. While ankyrin-B has been shown to interact with a variety of membrane and structural proteins located at different subcellular domains in cardiomyocytes, what regulates the specificity of ankyrin-B for particular interacting proteins remains elusive.

Methods and results

Here, we report the identification of two novel ankyrin-B isoforms AnkB-188 and AnkB-212 in human, rat, and mouse hearts. Novel cDNAs for both isoforms were isolated by long-range PCR of reverse-transcribed mRNA isolated from human ventricular tissue. The isoforms can be discriminated based on their function and subcellular distribution in cardiomyocytes. Heterologous overexpression of AnkB-188 increases sodium-calcium exchanger (NCX) membrane expression and current, while selective knockdown of AnkB-188 in cardiomyocytes reduces NCX expression and localization in addition to causing irregular contraction rhythms. Using an isoform-specific antibody, we demonstrate that the expression of AnkB-212 is restricted to striated muscles and is localized to the M-line of cardiomyocytes by interacting with obscurin. Selective knockdown of AnkB-212 significantly attenuates the expression of endogenous ankyrin-B at the M-line but does not disrupt NCX expression at transverse tubules in cardiomyocytes.

Conclusion

The identification and characterization of two functionally distinct ankyrin-B isoforms in heart provide compelling evidence that alternative splicing of the ANK2 gene regulates the fidelity of ankyrin-B interactions with proteins.

SUBMITTER: Wu HC

PROVIDER: S-EPMC4540146 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Publications

Identification and characterization of two ankyrin-B isoforms in mammalian heart.

Wu Henry C HC Yamankurt Gokay G Luo JiaLie J Subramaniam Janani J Hashmi Syed Shahrukh SS Hu Hongzhen H Cunha Shane R SR

Cardiovascular research 20150624 4

<h4>Aims</h4>Excitation-contraction coupling in cardiomyocytes requires the proper targeting and retention of membrane proteins to unique domains by adaptor proteins like ankyrin-B. While ankyrin-B has been shown to interact with a variety of membrane and structural proteins located at different subcellular domains in cardiomyocytes, what regulates the specificity of ankyrin-B for particular interacting proteins remains elusive.<h4>Methods and results</h4>Here, we report the identification of tw ...[more]

PMID: 26109584

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Project description:Serotonin (5-hydroxytryptamine, 5-HT) is an important neuroactive molecule, as neurotransmitters regulate various biological functions in vertebrates and invertebrates by binding and activating specific 5-HT receptors. The pharmacology and tissue distribution of 5-HT receptors have been investigated in several model insects, and these receptors are recognized as potential insecticide targets. However, little is known about the pharmacological characterization of the 5-HT receptors in important agricultural pests. In this study, we investigated the sequence, pharmacology, and tissue distribution of 5-HT7 receptors from oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae), an important migratory and polyphagous pest species. We found that the 5-HT7 receptor gene encodes two molecularly distinct transcripts, Msep5-HT7L and Msep5-HT7S, by the mechanism of alternative splicing in M. separata. Msep5-HT7S differs from Msep5-HT7L based on the deletion of 95 amino acids within the third intracellular loop. Two Msep5-HT7 receptor isoforms were activated by 5-HT and synthetic agonists α-methylserotonin, 8-hydroxy-DPAT, and 5-methoxytryptamine, resulting in increased intracellular cAMP levels in a dose-dependent manner, although these agonists showed much poorer potency and efficacy than 5-HT. The maximum efficacy of 5-HT compared to the two 5-HT isoforms was equivalent, but 5-HT exhibited 2.63-fold higher potency against the Msep5-HT7S than the Msep5-HT7L receptor. These two isoforms were also blocked by the non-selective antagonist methiothepin and the selective antagonists WAY-100635, ketanserin, SB-258719, and SB-269970. Moreover, two distinct mRNA transcripts were expressed preferentially in the brain and chemosensory organs of M. separata adults, as determined by qPCR assay. This study is the first comprehensive characterization of two splicing isoforms of 5-HT7 receptors in M. separata, and the first to demonstrate that alternative splicing is also the mechanism for producing multiple 5-HT7 isoforms in insects. Pharmacological and gene expression profiles offer important information that could facilitate further exploration of their function in the central nervous system and peripheral chemosensory organs, and may even contribute to the development of new selective pesticides.

Dataset Information

Identification and characterization of two ankyrin-B isoforms in mammalian heart.

Aims

Methods and results

Conclusion

Publications

Identification and characterization of two ankyrin-B isoforms in mammalian heart.

Similar Datasets

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