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A novel binding site between the voltage-dependent calcium channel Ca_V1.2 subunit and Ca_Vβ2 subunit discovered using a new analysis method for protein-protein interactions.

ABSTRACT: We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 subunit (Ca_V1.2) for the voltage-dependent calcium channel β2 subunit. Prokaryotic expression screening of the β2 subunit using an epitope library of Ca_V1.2 resulted in two overlapping clones of the C-terminal sequence of Ca_V1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of Ca_V1.2 and β2.

SUBMITTER: Murakami AM

PROVIDER: S-EPMC10460381 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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A novel binding site between the voltage-dependent calcium channel Ca<sub>V</sub>1.2 subunit and Ca<sub>V</sub>β2 subunit discovered using a new analysis method for protein-protein interactions.

Murakami Agnieszka M AM Nagatomo Katsuhiro K Miyoshi Ichro I Itagaki Shirou S Niwa Yasutaka Y Murakami Manabu M

Scientific reports 20230826 1

We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 ...[more]

PMID: 37634019

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Project description:BackgroundL-type Ca2+ channel CaV1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac CaV1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of CaV1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how CaV1.2 channels are alternatively spliced in diabetic heart remains unknown.MethodsDiabetic rat models were established by using high-fat diet in combination with low dose streptozotocin. Cardiac function and morphology were assessed by echocardiography and HE staining, respectively. Isolated neonatal rat ventricular myocytes (NRVMs) were used as a cell-based model. Cardiac CaV1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca2+ concentration was monitored by using Fluo-4 AM.ResultsWe find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased CaV1.2 channel with alternative exon 9* (CaV1.2E9*), but unchanged that with alternative exon 8/8a or exon 33. The splicing factor Rbfox2 expression is also increased in diabetic heart, presumably because of dominate-negative (DN) isoform. Unexpectedly, high glucose cannot induce the aberrant expressions of CaV1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates CaV1.2E9* channels proportion and downregulates Rbfox2 expression in NRVMs. By whole-cell patch clamp, we find GS application hyperpolarizes the current-voltage curve and window currents of cardiac CaV1.2 channels. Moreover, GS treatment raises K+-triggered intracellular Ca2+ concentration ([Ca2+]i), enlarges cell surface area of NRVMs and induces hypertrophic genes transcription. Consistently, siRNA-mediated knockdown of Rbfox2 in NRVMs upregulates CaV1.2E9* channel, shifts CaV1.2 window currents to hyperpolarization, increases [Ca2+]i and induces cardiomyocyte hypertrophy.ConclusionsAGEs, not glucose, dysregulates Rbfox2 which thereby increases CaV1.2E9* channels and hyperpolarizes channel window currents. These make the channels open at greater negative potentials and lead to increased [Ca2+]i in cardiomyocytes, and finally induce cardiomyocyte hypertrophy in diabetes. Our work elucidates the underlying mechanisms for CaV1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced CaV1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.

Dataset Information

A novel binding site between the voltage-dependent calcium channel Ca_V1.2 subunit and Ca_Vβ2 subunit discovered using a new analysis method for protein-protein interactions.

Publications

A novel binding site between the voltage-dependent calcium channel Ca<sub>V</sub>1.2 subunit and Ca<sub>V</sub>β2 subunit discovered using a new analysis method for protein-protein interactions.

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Dataset Information

A novel binding site between the voltage-dependent calcium channel CaV1.2 subunit and CaVβ2 subunit discovered using a new analysis method for protein-protein interactions.

Publications

A novel binding site between the voltage-dependent calcium channel Ca<sub>V</sub>1.2 subunit and Ca<sub>V</sub>β2 subunit discovered using a new analysis method for protein-protein interactions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

A novel binding site between the voltage-dependent calcium channel Ca_V1.2 subunit and Ca_Vβ2 subunit discovered using a new analysis method for protein-protein interactions.