Mechanosensitivity of the Bacterial Voltage-Gated Sodium Channel NaChBac

Abstract

INTRODUCTION: The voltage-gated sodium channel family spans from ancestral prokaryotic complexes found in bacterial extremophiles to eukaryotic proteins that are essential for excitability of most cell types. The SCN5A-encoded mechanosensitive voltage-gated channel Nav1.5 is expressed in electro-mechanical organs such as the heart and gastrointestinal tract. It is well-established that Nav1.5 is mechanosensitive, but biophysical studies of NaV mechanosensitivity are limited by the size of NaV proteins and their rapid kinetics, especially inactivation. Bacterial voltage-gated sodium channels recapitulate many of the eukaryotic NaV functional properties and have allowed significant advances in structural, functional and pharmacologic understanding of NaV channels and would serve as a good mechanosensitivity model. However, NaChBac mechanosensitivity has not been examined. AIMS: To determine if the bacterial Na+ channel NaChBac and a NaChBac non-inactivating mutant are mechanosensitive. METHODS: Nav1.5, NaChBac WT or the non-inactivating mutant T220A NaChBac constructs were co-transfected with GFP into HEK-293 cells. NaChBac mechanosensitivity was compared to Nav1.5 by whole Cell voltage-clamp with shear stress and in cell-attached patches using pressure clamp. RESULTS: Shear-stress increased Nav1.5 peak current by 18±3%. Similarly, shear-stress increases peak current in both WT NaChBac (+36.9%) and the non-inactivating mutant T220A (+41.6%). Clusters of single-channel openings (∼2 pS) were obtained from T220A in absence and presence of pressure (−30 mmHg). CONCLUSIONS: Effects of shear in peak current and voltage-dependency found in WT NaChBac and T220A were comparable to those found in Nav1.5. NaChBac T220A presented long-lasting frequent single channel openings, which were affected by pressure. NaChBac may serve as a good model to understand eukaryotic NaV channel mechanosensitivity.