EPITHELIAL NA+ CHANNEL-CYTOSKELETON INTERACTIONS
EPITHELIAL NA+ CHANNEL-CYTOSKELETON INTERACTIONS
批准号:
2145964
负责人:
PETER R. SMITH
金额:
$11.19万
依托单位国家:
美国
项目类别:
财政年份:
1994
资助国家:
美国
项目状态:
已结题
起止时间:
1994-01-01 至 1997-12-31
关键词:
Xenopus Xenopus oocyte animal tissue ankyrins apical membrane cellular polarity cytoskeleton epithelium fluorescent dye /probe human tissue immunocytochemistry intermolecular interaction intracellular transport laboratory mouse laboratory rabbit membrane structure renal tubular transport sodium channel sodium potassium exchanging ATPase spectrin tissue /cell culture
中文摘要
Na+重吸收肾上皮细胞含有Na+特异性通道,
在它们的顶膜中。 这些上皮Na+通道介导
在第一阶段,Na+从腔液进入细胞
产电性跨上皮Na+转运。 Na+的限制
通道顶端膜域是必不可少的载体
Na+穿过这些上皮的运输。 然而,机制
负责确定和维持Na+的分布
肾脏Na+重吸收的顶膜区域通道
上皮细胞目前尚不清楚。 初步实验表明
上皮Na+通道与细胞膜骨架相连,
蛋白质锚蛋白和血影蛋白。 据推测
Na+通道和细胞膜骨架之间的相互作用
参与维持Na+的极化分布
通道到顶膜。 因此,本建议的主要目标
研究的目的是探讨膜细胞骨架蛋白的作用,
锚蛋白和血影蛋白在决定和维持顶端细胞中的作用
钠重吸收肾上皮钠通道表面分布
上皮细胞 A6肾上皮细胞,一种来源于
非洲爪蟾的远端小管将被用作模型系统。 那里
有三个具体目标:(1)检验存在直接
上皮细胞Na+通道与细胞膜骨架的相互作用
蛋白质锚蛋白和血影蛋白,这种相互作用限制了Na+
顶膜微绒毛区域的通道
应用生物化学、免疫细胞化学和免疫细胞化学方法研究肾上皮细胞对Na+的重吸收。
荧光光漂白恢复(FPR)技术(2)
假设Na+通道-膜细胞骨架相互作用是
参与决定上皮Na+通道的分布,
Na+重吸收肾上皮细胞顶膜
非极化前体细胞向极化上皮细胞的分化
A6细胞(3)为了进一步验证Na+通道-
在特异性目的1中鉴定的膜细胞骨架复合物参与
在确定和维持Na+通道的分布中,
特定的膜结构域,非洲爪蟾卵母细胞表达系统将
用作体外模型。 这将通过注射卵母细胞来实现
带有编码上皮Na+通道的Poly(A)+RNA或合并感染
有义或反义的Na+通道Poly(A)+RNA
编码锚蛋白的寡核苷酸,随后进行免疫化学和FPR
分析表达渠道。 除了说明如何
确定这种必需通道蛋白的顶端分布,
维持在肾上皮中,本申请中提出的研究
这将增加我们对其他上皮离子通道
在它们各自的膜域中建立和维持。
此外,根据最近的证据表明,缺血性
损伤破坏了肾小管的膜细胞骨架,
维持不同的顶侧和基底侧膜域的能力,我们
研究将增加我们对缺血诱导的肾损伤的理解,
失败
英文摘要
Na+ reabsorbing renal epithelia contain Na+ specific channels situated
within their apical membranes. These epithelial Na+ channels mediate
entry of Na+ from the luminal fluid into the cell during the first stage
of electrogenic transepithelial Na+ transport. Restriction of the Na+
channels to the apical membrane domain is essential for the vectorial
transport of Na+ across these epithelia. However, the mechanisms
responsible for determining and maintaining the distribution of the Na+
channels to the apical membrane domains of renal Na+ reabsorbing
epithelia are presently not understood. Preliminary experiments suggest
that epithelial Na+ channels are linked to the membrane cytoskeletal
proteins ankyrin and spectrin in renal epithelia. It is hypothesized
that this interaction between Na+ channels and the membrane cytoskeleton
is involved in the maintenance of the polarized distribution of Na+
channels to the apical membrane. Thus, the primary goal of this proposed
study is to investigate the role of the membrane cytoskeletal proteins
ankyrin and spectrin in determining and maintaining the apical cell
surface distribution of epithelial Na+ channels in Na+ reabsorbing renal
epithelia. A6 renal epithelial cells, a cell line derived from the
distal tubule of Xenopus laevis will be used as the model system. There
are three specific aims:(1) To test the hypothesis that there is a direct
interaction between epithelial Na+ channels and the membrane cytoskeletal
proteins ankyrin and spectrin, and that this interaction restrict the Na+
channels to the microvillar domain of the apical membrane in
Na+reabsorbing renal epithelia using biochemical, immunocytochemical and
fluorescence photobleach recovery (FPR) techniques (2) To test the
hypothesis that Na+ channel-membrane cytoskeleton interactions are
involved in determining the distribution of epithelial Na+ channels to
the apical membrane of Na+ reabsorbing renal epithelia by examining
differentiation from unpolarized precursors to polarized epithelia cells
in A6 cells. (3) To further test the hypotheses that the Na+ channel-
membrane cytoskeleton complex identified in Specific Aim 1 is involved
in determining and maintaining the distribution of Na+ channels within
specific membrane domains, the Xenopus oocyte expression system will be
used as an in vitro model. This will be achieved by injecting oocytes
with Poly (A)+RNA coding for the epithelial Na+ channel or coinfection
of Na+ channel Poly (A)+RNA with either sense or antisense
oligonucleotides encoding ankyrin followed by immunochemical and FPR
analyses of expressed channels. In addition to elucidating how the
apical distribution of this essential channel protein is determined and
maintained in renal epithelia, the studies proposed in this application
will increase our understanding of how other epithelial ion channels are
established and maintained within their respective membrane domains.
Furthermore, in light of the recent evidence suggesting that ischemic
injury disrupts the membrane cytoskeleton of the renal tubules and their
ability to maintain distinct apical and basolateral membrane domains, our
studies will increase our understanding of ischemia induced renal
failure.
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批准号:10215712
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批准号:6517660
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UAB Cancer Research Experiences for Students
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批准号:9130759
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项目类别:
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资助金额:$28.4万
-
财政年份:1999
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依托单位:
UAB Cancer Research Experiences for Students
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批准号:9763450
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资助金额:$30.24万
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财政年份:1999
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负责人:PETER R. SMITH
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依托单位:
SODIUM CHANNEL APICAL PROTEIN XENOPUS (APX) INTERACTIONS
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批准号:6164589
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项目类别:
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资助金额:$18.37万
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财政年份:1999
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负责人:PETER R. SMITH
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依托单位:
SODIUM CHANNEL APICAL PROTEIN XENOPUS (APX) INTERACTIONS
-
批准号:6363051
-
项目类别:
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资助金额:$18.93万
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财政年份:1999
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负责人:PETER R. SMITH
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依托单位:
SODIUM CHANNEL APICAL PROTEIN XENOPUS (APX) INTERACTIONS
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批准号:6022004
-
项目类别:
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资助金额:$18.72万
-
财政年份:1999
-
负责人:PETER R. SMITH
-
依托单位:
EPITHELIAL NA+ CHANNEL-CYTOSKELETON INTERACTIONS
-
批准号:2145965
-
项目类别:
-
资助金额:$6.4万
-
财政年份:1994
-
负责人:PETER R. SMITH
-
依托单位:
EPITHELIAL SODIUM CHANNEL/CYTOSKELETON INTERACTIONS
-
批准号:2145967
-
项目类别:
-
资助金额:$11.58万
-
财政年份:1994
-
负责人:PETER R. SMITH
-
依托单位:
EPITHELIAL SODIUM CHANNEL/CYTOSKELETON INTERACTIONS
-
批准号:2145966
-
项目类别:
-
资助金额:$4.7万
-
财政年份:1994
-
负责人:PETER R. SMITH
-
依托单位:
EPITHELIAL SODIUM CHANNEL/CYTOSKELETON INTERACTIONS
-
批准号:2016679
-
项目类别:
-
资助金额:$12.04万
-
财政年份:1994
-
负责人:PETER R. SMITH
-
依托单位:
EPITHELIAL SODIUM CHANNEL/CYTOSKELETON INTERACTIONS
-
批准号:6032003
-
项目类别:
-
资助金额:$0.0万
-
财政年份:1994
-
负责人:PETER R. SMITH
-
依托单位:
海外基金