Method for screening a modulator of a TMEM16 family member

摘要

The present invention relates to a method for screening a modulator of a TMEM16 family member, which comprises the following steps:
(1) treating cells expressing the TMEM16 family member with a candidate of the modulator, and(2) determining whether the candidate alters distribution of a lipid selected from phosphatidylserine, phosphatidylcholine, and galactosylceramide in plasma membrane of the cells,

wherein a candidate which increases distribution of phosphatidylserine in the outer leaflet of plasma membrane compared to control is selected as a modulator enhancing a function of the TMEM16 family member, and a candidate which decreases distribution of phosphatidylserine in the outer leaflet of plasma membrane compared to control is selected as a modulator suppressing a function of the TMEM16 family member, and

a candidate which increases distribution of phosphatidylcholine or galactosylceramide in the inner leaflet of plasma membrane compared to control is selected as a modulator enhancing a function of the TMEM16 family member, and a candidate which decreases distribution of phosphatidylcholine or galactosylceramide in the inner leaflet of plasma membrane compared to control is selected as a modulator suppressing a function of the TMEM16 family member.

主权项

1. A method for screening an agent promoting or inhibiting a biological function of a transmembrane protein 16 (TMEM16) family member as a lipid scramblase, which comprises the following steps:
(1) treating TMEM16F deficient cells into which a gene encoding and expressing the TMEM16 family member selected from the group consisting of TMEM16C, TMEM16D, TMEM16G and TEMEM16J has been introduced with a candidate of the agent in the presence of Ca2+, and (2) determining whether the candidate alters distribution of a lipid selected from the group consisting of phosphatidylserine, phosphatidylcholine, and galactosylceramide in plasma membrane of the cells, wherein a candidate which significantly increases distribution of phosphatidylserine in the outer leaflet of plasma membrane compared to control is selected as an agent promoting a biological function of the TMEM16 family member as a lipid scramblase, and a candidate significantly which decreases distribution of phosphatidylserine in the outer leaflet of plasma membrane compared to control is selected as an agent inhibiting a biological function of the TMEM16 family member as a lipid scramblase, and a candidate which significantly increases distribution of phosphatidylcholine or galactosylceramide in the inner leaflet of plasma membrane compared to control is selected as an agent promoting a biological function of the TMEM16 family member as a lipid scramblase, and a candidate which significantly decreases distribution of phosphatidylcholine or galactosylceramide in the inner leaflet of plasma membrane compared to control is selected as an agent inhibiting a biological function of the TMEM16 family member as a lipid scramblase; and wherein the cells are human, monkey, mouse, or rabbit cells, the distribution of phosphatidylserine in plasma membrane is determined by detecting the binding between phosphatidylserine exposed to the cell surface and an agent having phosphatidylserine-binding property, and the distribution of phosphatidylcholine or galactosylceramide in plasma membrane is determined by utilizing a fluorescently-labeled lipid.