Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Poster Abstracts

1-POS Board 1 OPTICAL DEMONSTRATION THAT THE LIPID ENVIRONMENT IN THE PLASMA MEMBRANE INFLUENCES THE ACTIVITY OF A VOLTAGE SENSING DOMAIN

Haeun Lee ; Eugene Park 1 ; Bradley J. Baker 1 ; 1Korea Institute of Science and Technology, Seoul, South Korea 1 ; Nazarii Frankiv 1

Genetically encoded voltage indicators (GEVIs) convert membrane potential transients into an optical signal. The ArcLight family of GEVIs consists of a classic voltage sensing domain with four transmembrane segments (S1-S4) from the voltage sensing phosphatase gene family fused to a fluorescent protein. Positively charged arginines positioned every third amino acid in S4 cause a conformational change when the membrane potential is altered. The consensus charge distribution in S4 of the phosphatase gene family is R1-X-X-R2-X-X-I3-X-X-R4 (isoleucine occupies the R3 position). To investigate the role counter charges in S1-S3 play in determining the voltage response, we performed a lysine scan of the arginines in S4. The hypothesis was that the reduced hydrogen bond capability of lysine with only one nitrogen in its sidechain could act as a stop signal for further movement of S4. Lysines at the 2nd, 3rd, or 4th positions yielded uniform optical responses in the plasma membrane when expressed in HEK cells and subjected to whole cell voltage clamp. In contrast, the K1-X-X-R2-X-X-I3-X-X-R4 mutant despite uniform membrane expression, displayed a discontinuous voltage-dependent optical response in the plasma membrane suggesting that in some regions of the cell S4 could not respond to voltage. Voltage-dependent optical signals from the entire plasma membrane could be observed from the K1 mutant with the additional mutation of arginine at the I3 position (K1-X-X-R2-X X-R3-X-X-R4), however, different optical patterns were still detected in different regions of the plasma membrane. These results indicate that the varied lipid environment of the plasma membrane influences the conformation of the voltage sensing domain resulting in diverse functions of the protein. Other GEVI mutants exhibit similar behaviors indicating that GEVI activity may provide new insights into the effects membrane chemistry has on cell physiology.

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