On the mechanism of CFTR inhibition by a thiazolidinone derivative

Zoia Kopeikin, Yoshiro Sohma, Min Li, Tzyh Chang Hwang

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]- 5-[(4-carboxyphenyl)methylene]-2-thioxo- 4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitorinduced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

Original languageEnglish
Pages (from-to)659-671
Number of pages13
JournalJournal of General Physiology
Volume136
Issue number6
DOIs
Publication statusPublished - 2010 Dec

Fingerprint

Cystic Fibrosis Transmembrane Conductance Regulator
Adenosine Triphosphate
Rhodanine
Cricetulus
Inhibitory Concentration 50
Cations
Ovary
Hydrolysis
Membranes

ASJC Scopus subject areas

  • Physiology

Cite this

On the mechanism of CFTR inhibition by a thiazolidinone derivative. / Kopeikin, Zoia; Sohma, Yoshiro; Li, Min; Hwang, Tzyh Chang.

In: Journal of General Physiology, Vol. 136, No. 6, 12.2010, p. 659-671.

Research output: Contribution to journalArticle

Kopeikin, Zoia ; Sohma, Yoshiro ; Li, Min ; Hwang, Tzyh Chang. / On the mechanism of CFTR inhibition by a thiazolidinone derivative. In: Journal of General Physiology. 2010 ; Vol. 136, No. 6. pp. 659-671.
@article{9bf9ef178db545b39d5494fd3faee1cb,
title = "On the mechanism of CFTR inhibition by a thiazolidinone derivative",
abstract = "The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]- 5-[(4-carboxyphenyl)methylene]-2-thioxo- 4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitorinduced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels {"}inactivate{"} despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.",
author = "Zoia Kopeikin and Yoshiro Sohma and Min Li and Hwang, {Tzyh Chang}",
year = "2010",
month = "12",
doi = "10.1085/jgp.201010518",
language = "English",
volume = "136",
pages = "659--671",
journal = "Journal of General Physiology",
issn = "0022-1295",
publisher = "Rockefeller University Press",
number = "6",

}

TY - JOUR

T1 - On the mechanism of CFTR inhibition by a thiazolidinone derivative

AU - Kopeikin, Zoia

AU - Sohma, Yoshiro

AU - Li, Min

AU - Hwang, Tzyh Chang

PY - 2010/12

Y1 - 2010/12

N2 - The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]- 5-[(4-carboxyphenyl)methylene]-2-thioxo- 4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitorinduced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

AB - The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]- 5-[(4-carboxyphenyl)methylene]-2-thioxo- 4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitorinduced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

UR - http://www.scopus.com/inward/record.url?scp=78650045527&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78650045527&partnerID=8YFLogxK

U2 - 10.1085/jgp.201010518

DO - 10.1085/jgp.201010518

M3 - Article

C2 - 21078867

AN - SCOPUS:78650045527

VL - 136

SP - 659

EP - 671

JO - Journal of General Physiology

JF - Journal of General Physiology

SN - 0022-1295

IS - 6

ER -