GATING MECHANISMS OF RETINAL ROD CGMP ACTIVATED CHANNELS
GATING MECHANISMS OF RETINAL ROD CGMP ACTIVATED CHANNELS
批准号:
6342623
负责人:
William N Zagotta
金额:
$16.08万
依托单位:
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 2003-12-31
关键词:
SDS polyacrylamide gel electrophoresis Xenopus Xenopus oocyte allosteric site alternatives to animals in research aminoacid binding proteins binding sites conformation cyclic GMP electrophysiology membrane channels molecular cloning phosphorylation polymerase chain reaction protein protein interaction protein structure function rod cell single cell analysis site directed mutagenesis surface plasmon resonance visual photoreceptor visual phototransduction voltage /patch clamp yeast two hybrid system
中文摘要
环核苷酸门控 (CNG) 离子通道在多个方面发挥着关键作用
视网膜信号处理的阶段。在感光器外层
它们检测并发出 cGMP 浓度下降信号
由于视紫红质吸收光而产生;在内段
在视锥细胞上,它们调节发射机释放到
双极细胞;在单双极细胞中,它们负责
对谷氨酸的抑制反应。 CNG渠道高度专业化
以表彰它们在信号处理中的作用。我们研究的长期目标
is to understand the molecular mechanisms that underlie their
专业化。 cGMP 通过直接结合到
每个的羧基末端区域都有环核苷酸结合域
通道子单元。 cAMP 是这些通道的非常差的激活剂。这个
环核苷酸的选择性来自于构象的变化
与离子打开相关的环核苷酸结合位点
导电孔。此外,这些通道由 Ca/2 调制 -
钙调蛋白和磷酸化。这种调制被认为涉及
每个氨基酸末端区域之间的蛋白质-蛋白质相互作用
亚基和环核苷酸结合结构域。在这个提案中我们
将检查构象变化背后的分子机制
在环核苷酸结合位点以及它是如何受其调节的
蛋白质-蛋白质相互作用。渠道将被研究
通过在电生理学中外源表达 cDNA 克隆
非洲爪蟾卵母细胞,通过表达氨基末端进行生化分析
区域和通道的环核苷酸结合域融合
细菌中的蛋白质。我们将探究构象变化
具有单通道记录的环核苷酸结合位点,位点
directed mutagenesis, and state-dependent cysteine modification.在
此外,使用蛋白质相互作用测定,我们将检查
structural determinants for the interaction between the amino-terminal
区域和环核苷酸结合域以及它们如何解释
Ca/2-钙调蛋白和磷酸化对通道的调节。
这些实验应该可以深入了解 CNG 通道的作用
在信号转导方面。
英文摘要
Cyclic nucleotide-gated (CNG) ion channels play a key role at several
stages of signal processing in the retina. In photoreceptor outer
segments, they detect and signal the drop in cGMP concentration
resulting from the absorption of light by rhodopsin; in inner segments
on the cone photoreceptors they modulate transmitter release onto the
bipolar cells; and in one-bipolar cells they are responsible for the
inhibitory response to glutamate. CNG channels are highly specialized
for their role in signal processing. The long-term goal of our research
is to understand the molecular mechanisms that underlie their
specializations. cGMP activates the channel by binding directly to a
cyclic nucleotide-binding domain in the carboxyl-terminal region of each
channel subunit. cAMP is a very poor activator of these channels. This
cyclic nucleotide selectivity arises from a conformational change in the
cyclic nucleotide-binding site associated with opening of the ion
conducting pore. Furthermore, these channels are modulated by Ca/2+-
calmodulin and phosphorylation. This modulation is thought to involve a
protein-protein interaction between the amino-terminal region of each
subunit and the cyclic nucleotide-binding domain. In this proposal we
will examine the molecular mechanism underlying the conformation change
in the cyclic nucleotide-binding site and how it is regulated by this
protein-protein interaction. The channels will be studied
electrophysiologically by exogenously expressing the cDNA clones in
Xenopus oocytes, and biochemically by expressing the amino terminal
region and cyclic nucleotide-binding domain of the channel as fusion
proteins in bacteria. We will probe the conformation change in the
cyclic nucleotide-binding site with single-channel recording, site-
directed mutagenesis, and state-dependent cysteine modification. In
addition, using protein interaction assays, we will examine the
structural determinants for the interaction between the amino-terminal
region and the cyclic nucleotide-binding domain and how they account for
modulation of the channels by Ca/2+-calmodulin and phosphorylation.
These experiments should provide insight into the role of CNG channels
in signal transduction.
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海外基金