The role of VAMP proteins in K+ channel regulation

Zhang, Ben (2015) The role of VAMP proteins in K+ channel regulation. PhD thesis, University of Glasgow.

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SNARE (soluble N-ethylmaleimide-sensitive factor protein
attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis and pathogen defense. A subset of SNAREs, including the Arabidopsis SNARE SYP121, are known also to coordinate solute uptake via physical
interactions with K+ channels and to moderate their gating at the plasma membrane.
R-SNAREs, also known as vesicle-associated membrane proteins (VAMPs), are most commonly associated with trafficking vesicles. In guard cells of Arabidopsis, ABA-dependent stomatal closure was inhibited when the expression of VAMP71 family genes were suppressed
by an antisense VAMP711 construct. Such results reveal that VAMPs play an important role in plant response to stress, especially in the regulation of stomatal closure under ABA treatment. Two R-SNAREs, VAMP721 and VAMP722, are known to assemble in SNARE core complexes with SYP121 and with its closest homolog SYP122, which raises the question whether the channel interaction might extend to the R-SNAREs leading to VAMP regulating channel gating.
To answer this question, I investigated the interaction between the VAMP7 proteins with KC1 and KAT1 K+ channels by mating based Split-Ubiquitin System (mbSUS) assay, and verified these interactions by ratiometric bimolecular fluorescence complementation (rBiFC) assay. I found VAMP721 and VAMP722, but not VAMP723, interacted with the
channels. The selective binding was associated with the VAMP longin domain, notably with Tyr57. What is the effect of the VAMP-K+ channel interaction on transmembrane ion transport and vesicle traffic? I found VAMP721 affected K+ channel gating and suppressed the K+ current within the physiological voltage range by electrophysiological analysis in Xenopus oocytes and in VAMP over-expression wild type Arabidopsis. The effect
of VAMP721 on K+ channel regulation was opposite to the action of SYP121 on K+ channel. From localization, interaction and electrophysiological studies, I was able to show that Tyr57 is a key residue required both for VAMP-dependent gating of the K channels and for localization of the VAMPs at the plasma membrane. For vesicle traffic, I found overexpression of full length VAMP721 in Arabidopsis seedlings blocked the secretion of secYFP while coexpressing with KC1 K+ channel rescued the traffic block, demonstrating a potential action of VAMP-K+ channel interaction on secretory traffic. These results add to understanding the interaction between SNARE and K+ channels that link membrane traffic with osmotically active solute transport in the plant.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > Q Science (General)
Q Science > QK Botany
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Blatt, Professor Michael
Date of Award: 2015
Depositing User: Dr Ben Zhang
Unique ID: glathesis:2015-6230
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 31 Mar 2015 08:44
Last Modified: 30 Mar 2018 11:07

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