TY  - JOUR
A1  - Pandey, Yogesh
T1  - Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5.
JF  - Biochimica et Biophysica Acta (BBA) - Biomembranes
N2  - Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.
KW  - Cell-free protein synthesis
KW  - Electrophysiology
KW  - Membrane proteins
KW  - Micro-translantation
KW  - Protein expression
Y1  - 2023
U6  - https://doi.org/10.1016/j.bbamem.2023.184144
SN  - 1879-2642
SN  - 0005-2736
VL  - 1865
IS  - 5
PB  - Elsevier
CY  - Amsterdam
ER  -