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 β-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 β-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 HABC 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 (τ=12.3±0.8 ms) much more than (P<0.05) WT Syn-1A (τ=7.9±0.8 ms; control τ=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 (V1/2=33.1±1.3 and −29.4±1.1 mV respectively; control V1/2=−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.
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Research Article|
March 22 2005
Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels
Yuk M. LEUNG;
Yuk M. LEUNG
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
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Youhou KANG;
Youhou KANG
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
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Fuzhen XIA;
Fuzhen XIA
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
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Laura SHEU;
Laura SHEU
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
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Xiaodong GAO;
Xiaodong GAO
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
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Huanli XIE;
Huanli XIE
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
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Robert G. TSUSHIMA;
Robert G. TSUSHIMA
1
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
1Correspondence may be addressed to either of the authors (email herbert.gaisano@utoronto.ca and r.tsushima@utoronto.ca).
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Herbert Y. GAISANO
Herbert Y. GAISANO
1
1Departments of Medicine and Physiology, University of Toronto, Toronto, Canada M5S 1A8
1Correspondence may be addressed to either of the authors (email herbert.gaisano@utoronto.ca and r.tsushima@utoronto.ca).
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Publisher: Portland Press Ltd
Received:
September 22 2004
Revision Received:
October 27 2004
Accepted:
November 01 2004
Accepted Manuscript online:
November 01 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 387 (1): 195–202.
Article history
Received:
September 22 2004
Revision Received:
October 27 2004
Accepted:
November 01 2004
Accepted Manuscript online:
November 01 2004
Citation
Yuk M. LEUNG, Youhou KANG, Fuzhen XIA, Laura SHEU, Xiaodong GAO, Huanli XIE, Robert G. TSUSHIMA, Herbert Y. GAISANO; Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels. Biochem J 1 April 2005; 387 (1): 195–202. doi: https://doi.org/10.1042/BJ20041625
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