G009 Regulation of the cardiac sodium channel Nav1.5 by a member of MAGUK proteins: SAP97

Abstract

Nav1.5 is the voltage-gated sodium channel that initiates the cardiac action potential. A precise regulation and localisation of Nav1.5 channels in cardiomyocytes is thus necessary for correct heart function. The three last amino-acids of Nav1.5 (SIV) constitute a PDZ-domain binding motif known to interact with the syntrophin-dystrophin complex and PDZ domains found in proteins of the MAGUK family. Among their multiple roles, MAGUK proteins can cluster proteins and localize them at the plasma membrane. We investigated the interaction between SAP97, one cardiac MAGUK protein, and Nav1.5. We postulated that this interaction may be implicated in correct localisation, anchoring, turn-over and/or regulation of Nav1.5 biophysical properties. Pull-down experiments performed with Nav1.5 C-terminus fusion proteins and human or mouse heart protein extracts revealed that the association between SAP97 and Nav1.5 depends on the PDZ-domain binding motif of Nav1.5. This interaction was specific for SAP97 and Nav1.5 as no pull-down could be detected with PSD95 or ZO-1, two MAGUK proteins also expressed in human heart. The functional consequences of this interaction were studied via patch-clamp experiments. Silencing of SAP97 reduced the whole-cell sodium current measured in HEK293 cells stably expressing Nav1.5 channels without decreasing the total protein amount. In control or silenced HEK293 cells, sodium current produced by Nav1.5 δSIV channels was reduced compared to WT. Immunostainings on frozen mouse heart slices demonstrated the colocalisation of Nav1.5 and dystrophin specifically at lateral membranes, but not at the intercalated discs. The possible colocalisation of Nav1.5 and SAP97 at the level of intercalated disks is currently investigated. This would support the hypothesis of the presence of two pools of Nav1.5 channels: one targeted at lateral membranes by the syntrophin-dystrophin complex, and another one targeted at intercalated discs by SAP97. These findings strongly support the existence of an interaction between Nav1.5 and SAP97 in cardiac tissue. This interaction also depends on the presence of Nav1.5 PDZ-domain binding motif and may play a role in determining the channel density at the plasma membrane. Additional biochemistry, cytochemistry and biophysical experiments will allow us to further address this question.