2013 Fiscal Year Research-status Report
Investigation of the dynamic rearrangements of the pore structure of P2X2 receptor channel upon voltage dependent activation
| Project/Area Number |
25460307
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Research Institution | National Institute for Physiological Sciences |
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Principal Investigator |
KECELI Batu 生理学研究所, 分子生理研究系, 特別協力研究員 (80645250)
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| Project Period (FY) |
2013-04-01 – 2016-03-31
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| Keywords | ligand gated channel / voltage dependent gating / SCAM / mutagenesis / VCF / ATP |
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| Research Abstract |
For SCAM and VCF approaches to be used in this study we prepared cysteine substituted constructs for TM1 and TM2 helices, over which native cysteine residues were mutated to a neutral threonine mutation (3T-WT). Background construct 3T-WT showed similar voltage dependent gating properties with WT P2X2. Over 3T-WT constructs we introduced one cysteine at each time from L29 to Q52 for TM1 (14) and from I328 to I351 (24) for TM2. In summary over 3T-WT background we made 38 different constructs for our experiments and identified that most of those constructs were functional.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Before starting the detailed analyzes of SCAM and VCF in various conditions for our constructs, it was necessary to confirm that our background construct 3T-WT was functioning properly. Moreover, cysteine mutants over 3T-WT were checked for their functionality and results were positive. Up to this time, by confirming the proper functionality of our constructs, we believe we can safely move for the detailed electrophysiology and fluorometry analyzes of our constructs, as we planned.
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| Strategy for Future Research Activity |
In the next step using our well confirmed functional constructs, we plan to analyze the TM domains' conformational rearrangements in voltage -and [ATP]-dependent gating using SCAM and VCF techniques. Depending on the interpretation of the results, if necessary we also plan FRET experiments which will enhance our understanding of the detailed conformational rearrangements of TM domains with respect to each other in the voltage -and [ATP]-dependent gating.
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| Expenditure Plans for the Next FY Research Funding |
We plan to make site directed cysteine accessibility analyses and voltage clamp fluorometry experiments for each one of the 38 constructs, in the absence/presence of ATP in the hyperpolarized and depolarized conditions, which requires an extensive usage of thiol modifying chemical reagents and fluorescent labeled cysteine tethering molecules. In case of promising results we also expect to perform FRET analyses which will require further expenditure in terms of novel construct design. Considering the fact that we will present our results in national and/or overseas scientific meetings, we plan to use the incurring amount to be used in the next fiscal year.
Incurring amount will be used for; (1) Purchasing chemicals for SCAM and VCF experiments, (2) Purchasing consumables for electrophysiology and molecular biology experiments, (3) Tavel expenses for the National and International scientific meetings, (4) Publication expenses for the peer reviewed journals.
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