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Distribution, isolation and reconstitution of functional molecules related to the taste-signal transduction.

与味觉信号转导相关的功能分子的分布、分离和重建。

基本信息

批准号：
04557080
负责人：
SATO Toshihide
金额：
$ 5.89万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

(A) Experiments with isolated taste cells :(1) We examined the characteristics and the spacial distribution of three ion channels on the frog taste cell membrane contributing to the salt-signal transduction in detail : (a) 80 pS Ca^<2+> -dependent K^+ channels. (b) 40 pS delayd-rectifying K^+ channels blocked by cAMP-dependent protein kinase. (c) 30 pS Ca^<2+>-dependent nonselective cation channels (NSC).(2) The distribution density of K^+ channels was much higher at the receptive membrane area than at the other portion.(3) NSC activity was observed uniformly thoughout the plasma membrane.(B) Experiments with artificial membranes :(1) In the experiments where channel proteins extracted from homogenates of the frog taste cell membrane were incorporated into an artificial membrane using the tip-dip method, we observed activity of channels having conductance similar to those obtained in (A).(2) It was difficult that channel proteins from taste cells were stably and readily employed as a taste sensor for a long period at the room temperature. Therefore, we used various ionophores instead of channel proteins.(3) We used valinomycin as K^+ channel analogue, and monencin, novobiocin, gramicidin, etc. as NSC analogue.(4) Mixtures of these ionophores were incorporated into a membrane of polyvinyl chloride (PVC), where di-n-octyl phtalate (FADO) and tetrahydrofuran (THF) were used as a plasticizer and a solvent respectively, and the responsivities to various salt solutions were examined.(5) Various combinations of different ionophores reconstituted into PVC were found to potentiate the ability to discriminate various salt stimuli, and then enables us to simulate the real salt responses in the taste cells. These results suggest that a sensor kit consisting of ionophores, PVC,FADO and THF can be developed in future.

(A)离体味觉细胞实验：（1）我们详细研究了青蛙味觉细胞膜上参与盐信号传导的三种离子通道的特征和空间分布：（a）80 pS Ca^<2+>依赖性K^+通道。(b)cAMP依赖性蛋白激酶阻断的40 pS延迟整流钾通道。(c)30 pS Ca^2+依赖性非选择性阳离子通道（NSC）。(2)感受膜区的K^+通道分布密度明显高于其他部位。(3)NSC活性在整个质膜上呈均匀分布。(B)人工膜的实验：（1）在使用尖端-浸渍法将从青蛙味觉细胞膜的匀浆中提取的通道蛋白掺入人工膜的实验中，我们观察到具有与（A）中获得的那些类似的电导的通道的活性。(2)来自味觉细胞的通道蛋白很难在室温下长时间稳定和容易地用作味觉传感器。因此，我们使用各种离子载体代替通道蛋白。(3)我们用缬氨霉素作为K^+通道类似物，用莫能菌素、新生霉素、短杆菌肽等作为NSC类似物。(4)这些离子载体的混合物被纳入到聚氯乙烯（PVC）的膜，其中邻苯二甲酸二正辛酯（FADO）和四氢呋喃（THF）分别用作增塑剂和溶剂，和各种盐溶液的响应进行了检查。(5)不同的离子载体重组到PVC的各种组合被发现，以加强区分各种盐刺激的能力，然后使我们能够模拟味觉细胞中的真实的盐反应。这些结果表明，由离子载体，PVC，FADO和THF组成的传感器套件可以在未来开发。