Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.643115
Title: The role of VAMP proteins in K+ channel regulation
Author: Zhang, Ben
ISNI:       0000 0004 5354 1070
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2015
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Abstract:
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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.643115  DOI: Not available
Keywords: Q Science (General) ; QK Botany
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