title: Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels.
abstract: 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.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Canada M5S 1A8.
authors.forename: Yuk M
authors.initials: YM
authors.lastname: Leung
authors.forename: Youhou
authors.initials: Y
authors.lastname: Kang
authors.forename: Fuzhen
authors.initials: F
authors.lastname: Xia
authors.forename: Laura
authors.initials: L
authors.lastname: Sheu
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Gao
authors.forename: Huanli
authors.initials: H
authors.lastname: Xie
authors.forename: Robert G
authors.initials: RG
authors.lastname: Tsushima
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D000954:Antigens, Surface / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D051668:Delayed Rectifier Potassium Channels; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D024642:Potassium Channels, Voltage-Gated / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary / Q000502:physiology; D051669:Shab Potassium Channels; D050827:Syntaxin 1; D014162:Transfection / Q000379:methods
chemical_list: D000954:Antigens, Surface; D051668:Delayed Rectifier Potassium Channels; C496988:KCNB1 protein, human; D009419:Nerve Tissue Proteins; D024642:Potassium Channels, Voltage-Gated; C494212:STX1A protein, human; D051669:Shab Potassium Channels; D050827:Syntaxin 1