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Journals / Journal of Neurological Sciences (Turkish) / 2006 / Cilt: 23 - Sayı: 4

Determination of functional regions of human DAT for labeling with fluorescent proteins for further use in fret studies

İnsan dopamin taşıyıcısında fret çalışmalarında kullanabilmek amacıyla floresan proteinlerle işaretlenebilecek işlevsel bölgelerinin belirlenmesi

Oya ORUN
Pages
287–294
Publication Date
2022-10-19
DOI
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AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

Linker sequences attached to the terminals of eCFP did not change this pattern.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:The dopamine transporter tightly regulates dopamine neurotransmission by the rapid uptake of released dopamine back into the presynaptic nerve terminal.
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Key Findings & Data:This study aimed to determine functional positions for eCFP insertion into the human dopamine transporter, a membrane protein with 12 transmembrane domains. eCFP has been inserted into the N-terminus of human DAT and at positions 515 and 587.
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Conclusion & Impact:Linker sequences attached to the terminals of eCFP did not change this pattern.
🔬Design:Research Article
📍Scope:Comprehensive Sample
⚙️Variables:Domain Parameters

Abstract

The dopamine transporter tightly regulates dopamine neurotransmission by the rapid uptake of released dopamine back into the presynaptic nerve terminal. This study aimed to determine functional positions for eCFP insertion into the human dopamine transporter, a membrane protein with 12 transmembrane domains. eCFP has been inserted into the N-terminus of human DAT and at positions 515 and 587. This will reveal available regions for further FRET analyses. Uptake assays showed that only the N-terminus is functional, while the other two positions exhibit extremely low rate of transport, even though their affinities are close to the wild type. These results were supported by surface expression profile observed on the microscope. Linker sequences attached to the terminals of eCFP did not change this pattern.

Özet

Dopamin taşıyıcıları presinaptik uçtan salınan dopaminin sinaptik boşluktan geri alınarak (uptake) dopaminerjik sinyalin sonlandırılmasında çok önemli rol oynarlar. Bu çalışmada 12 zarı kateden bölge içeren bir zar proteini olan insan dopamin taşıyıcısının çeşitli bölgelerine eCFP proteininin yerleştirilmesi ve etiketli proteinlerin işlevsel durumunun saptanması amaçlandı. eCFP proteini, insan DAT proteinin N-ucu bölgesine ve 515 ve 587.amino asitlerin bulunduğu bölgelere takıldı. Böylelikle, bir sonraki aşamada gerçekleştirilecek FRET analizlerinde kullanılabilecek pozisyonlar belirlenmiş olacaktır. Gerialım testleri yalnızca N-ucu bölgesinin işlevsel olduğunu, p515 ve p587 bölgelerinin ise yaban tiple benzer ilginlikte olmalarına rağmen geri alım hızlarının çok düşük olduğunu gösterdi. Bu sonuç mikroskopla elde edilen yüzey ekspresyonu sonuçları ile de doğrulandı. eCFP proteinine eklenen esnek bağlaç dizileri de bu sonucu değiştirmedi.

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Cite This Article

ORUN, O. (2022). Determination of functional regions of human DAT for labeling with fluorescent proteins for further use in fret studies. Journal of Neurological Sciences (Turkish), Cilt: 23 - Sayı: 4, 287–294. https://ajindex.com/articles/562805

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