Journal Article

Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc

Esperanza Agullo-Pascual, Xianming Lin, Alejandra Leo-Macias, Mingliang Zhang, Feng-Xia Liang, Zhen Li, Anna Pfenniger, Indra Lübkemeier, Sarah Keegan, David Fenyö, Klaus Willecke, Eli Rothenberg and Mario Delmar

in Cardiovascular Research

Published on behalf of European Society of Cardiology

Volume 104, issue 2, pages 371-381
Published in print November 2014 | ISSN: 0008-6363
Published online November 2014 | e-ISSN: 1755-3245 | DOI: https://dx.doi.org/10.1093/cvr/cvu195
Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc

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Aims

It is well known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein-interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant ‘Cx43D378stop’) led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell–cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43–INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: (i) the morphology of clusters formed by the microtubule plus-end tracking protein ‘end-binding 1’ (EB1), (ii) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin-rich sites, and (iii) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa.

Methods and results

Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa, and microtubules.

Conclusions

NaV1.5 and EB1 localization at the cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.

Keywords: Cx43; NaV1.5; EB1; Microtubule; Area composita; Connexome

Journal Article.  6229 words.  Illustrated.

Subjects: Cardiovascular Medicine

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