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

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 (I-Na) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-I-Na 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 Na(V)1.5, 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 I-Na. Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and Na(V)1.5 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 I-Na exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, I-Na, and microtubules. Na(V)1.5 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.