Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels

• Published in: Biochemical journal Vol. 387; no. Pt 1; pp. 195 - 202
• Main Authors: Leung, Yuk M, Kang, Youhou, Xia, Fuzhen, Sheu, Laura, Gao, Xiaodong, Xie, Huanli, Tsushima, Robert G, Gaisano, Herbert Y
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 01.04.2005
• Subjects: Antigens, Surface - biosynthesis; Antigens, Surface - chemistry; Antigens, Surface - physiology; Delayed Rectifier Potassium Channels; Humans; Ion Channel Gating - physiology; Kidney - chemistry; Kidney - cytology; Kidney - embryology; Kidney - metabolism; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - physiology; Patch-Clamp Techniques - methods; Potassium Channels, Voltage-Gated - biosynthesis; Potassium Channels, Voltage-Gated - genetics; Potassium Channels, Voltage-Gated - metabolism; Protein Structure, Quaternary - physiology; Shab Potassium Channels; Syntaxin 1; Transfection - methods
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20041625

We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.