Binding of Syntaxin 1A to the C-terminus of hERG Channels Affects Channel Trafficking and Inactivation

Abstract

The SNARE protein, syntaxin 1A (STX1A), functionally regulates cardiac ion channels, including the human ether-a-go-go related gene (hERG) which encodes the pore-forming voltage-gated K+ channel underlying IKr in the heart. The primary mode for STX1A-dependent inhibition of hERG channel function is trafficking impairment which can be rescued by reduced temperature or the high-affinity channel blocker, E4031. A secondary mode is achieved by the production of a hyperpolarizing shift in the voltage dependence of steady-state inactivation. Here we report the STX1A binding region on hERG channels. GST pulldown and coimmunoprecipitation demonstrates that the cytosolic SNARE motif-containing STX1A-H3 domain preferentially binds to hERG. This cytosolic domain is attached to the TM region by a short inflexible linker and is 60 residues in length. Use of N- (hERG-Δ2-16 and hERG-Δ2-354) and C-terminal truncation mutations (hERG-Δ1120, hERG-Δ1045, hERG-Δ1000, hERG-Δ960, hERG-Δ899, hERG-Δ860-899, hERG-Δ860, and hERG-Δ814) demonstrates that STX1A binds to all truncation mutations tested. The hERG C-terminus begins at approximately residue 670 immediately adjacent to the cytosolic portion of the S6 helices. Therefore, we deduce that the STX1A-H3 domain interacts with the C-terminus of hERG channels between residues 670 and 814. Functional analysis of C-terminal truncation mutations demonstrates that STX1A inhibits the trafficking of truncations up to hERG-Δ1000, but has no effect on hERG-Δ960 while enhancing trafficking and function of hERG-Δ899. We infer that STX1A-interaction disrupts normal protein folding thereby inhibiting channel trafficking, and may alter the movement of S6 helices affecting the hERG channel inactivation gate, shifting the voltage-sensitivity of channel inactivation. SNARE protein-mediated regulation of cardiac ion channels represents a novel biological mechanism that may have universally intrinsic implications for normal and diseased heart function.(Supported by HSFO)