A Novel Noncanonical Role of Cx43 in the Heart: Ensuring the Arrival of Nav1.5 to the Intercalated Disk

Abstract

Background In its classic description, the function of connexins is to form gap junctions. Although this description stands, recent studies show that connexin43 (Cx43) is not only a pore-forming gap junction protein. In fact, Cx43 is a part of a protein interacting network (the connexome), which likely regulates other functions in a gap junction–independent manner. Recently, it was reported that loss of the last five C-terminal amino acids of Cx43 (Cx43D378stop) leads to lethal ventricular arrhythmias in mice. Interestingly, the mutated Cx43 protein localized at the intercalated disk (ID), and gap junction channels showed normal properties. However, whole-cell sodium current (I Na ) density was decreased. Here, we explored the mechanisms relating Cx43 to I Na in ventricular myocytes. Methods and Results Functional assays: Adult myocytes from Cx43D378stop and Cre-negative (control) mice were tested by cell-attached macropatch, scanning ion conductance microscopy, and superresolution scanning patch clamp. We observed that Cx43D378stop caused a selective reduction in the number of functional sodium channels exclusively at the ID pool. Superresolution fluorescence microscopy: Two-color direct stochastic optical reconstruction microscopy (20-nm resolution) showed Na v 1.5 clusters in close proximity (or overlapping) to N-cadherin plaques. The distance between Na v 1.5 clusters and the cell end increased from 57.2 ± 12 nm (n = 365) in control to 111.7 ± 11 nm n=446) in Cx43D378stop myocytes ( P v 1.5 surface expression. This coincided with separation of the microtubule plus-end protein EB1 from the N-cadherin–rich cell end, from 23.7 ± 31.9 nm (n = 665) in control to 123.5 ± 13.5 nm (n = 502) in Cx43D378stop cells ( P P Conclusions Functional surface expression of Na v 1.5 at the ID depends on preservation of the Cx43 C-end. We propose that Cx43 is necessary for proper arrival of the microtubule plus-end to the ID and delivery of its cargo, including Na v 1.5. These gap junction–independent functions of Cx43 are vital to proper electrical function.