2007 Fiscal Year Final Research Report Summary
Study on the physiological function and the modulation of neuronal Ca^<2+> channels
| Project/Area Number |
18500306
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Tokyo Metropolitan Organization for Medical Research |
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Principal Investigator |
NUKADA Toshihide Tokyo Metropolitan Organization for Medical Research, Tokyo Metropolitan Origanazation for Medical Research,Tokyo Institute of Psychiatry, Head (80189349)
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| Project Period (FY) |
2006 – 2007
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| Keywords | Signal Transduction / Voltage-dependent Ca^<2+> Channel / Single-molecule / Imaging / Fluorescent labelling / TIRFM / Channel Opening and Closing / Epitope Tag |
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| Research Abstract |
A channel gating model has been proposed from the crystal structure analysis of voltage-sensitive K+ channels, in which, in response to depolarization, an S3b-S4a "paddle" may move relative to the rest of the voltage sensor domain. Aiming to monitor the voltage-sensing conformational change of single ion channels, a myc-epitope tag was, therefore, attached to the α1 subunits (α1B and α1A) of N- and P/Q-type voltage-dependent Ca^<2+> channels in the internal or external end of S4 segment of the repeat I, II, III or IV. Among these 16 kinds of mutant α1B and α1A, three myc-tagging mutants showed activities and kinetics similar to those of the wild-type channels, when expressed as the α1α2/δ1β1a combination in Xenopus oocytes. Total internal reflection fluorescence microscopy (TIRFM) was, then, used to image near- membrane cytosolic Ca^<2+> signals through single mutant channels by injection of Fluo-4-dextran as a Ca^<2+> indicator. At resting potentials, the fluorescence was usually stationary in the oocytes expressing these three mutant channels. By contrast, depolarization of the oocytes resulted in the sporadic appearance of numerous, transient bright spots in the TIRFM image. These indicate that the sparklets arise from Ca^<2+> flux through individual myc-tagging channels. In order to detect a single Ca^<2+> channel in the plasma membrane, the myc-tag on the mutant α1's was labeled by an anti-myc-epitope antibody conjugated with Alexa Fluor 488. Rapid movement of bright spots was detectable only in oocytes expressing the mutant channels, as observed in mammalian cultured cells expressing N-type channels fused with GFP at the α1B C-terminus. Moreover, the intensity of these spots was influenced by the depolarization of oocytes. From the present results, changes in the fluorescence intensity can be used as an indicator of depolarization-induced local protein motion of the α1subunit of voltage-dependent Ca^<2+> channel.
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[Journal Article] Site-directed mutagenesis.2007
Author(s)
Yamamoto H, et. al.
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Journal Title
In : Sigma receptors : chemistry, cell biology and clinical implications (Matsumoto RR, Bowen WD, Su TP, eds) New York : Springer
Pages: 113-125
Description
「研究成果報告書概要(欧文)」より
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