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Titolo:
CFTR: Covalent modification of cysteine-substituted channels expressed in Xenopus oocytes shows that activation is due to the opening of channels resident in the plasma membrane
Autore:
Liu, XH; Smith, SS; Sun, F; Dawson, DC;
Indirizzi:
Oregon Hlth Sci Univ, Dept Physiol Pharmacol, Portland, OR 97201 USA Oregon Hlth Sci Univ Portland OR USA 97201 rmacol, Portland, OR 97201 USA Univ Michigan, Dept Physiol, Ann Arbor, MI 48109 USA Univ Michigan Ann Arbor MI USA 48109 ept Physiol, Ann Arbor, MI 48109 USA
Titolo Testata:
JOURNAL OF GENERAL PHYSIOLOGY
fascicolo: 4, volume: 118, anno: 2001,
pagine: 433 - 446
SICI:
0022-1295(200110)118:4<433:CCMOCC>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
TRANSMEMBRANE CONDUCTANCE REGULATOR; CYSTIC-FIBROSIS GENE; NUCLEOTIDE-BINDING DOMAINS; EPITOPE-TAGGED CFTR; CHLORIDE CHANNELS; CL CHANNELS; SODIUM-CHANNELS; ATP HYDROLYSIS; CELL-SURFACE; CHO CELLS;
Keywords:
trafficking; MTS reagents; labeling; mutagenesis; oocyte expression;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
54
Recensione:
Indirizzi per estratti:
Indirizzo: Dawson, DC Oregon Hlth Sci Univ, Dept Physiol Pharmacol, L334,3181 SW Sam Jackson Pk Rd, Portland, OR 97201 USA Oregon Hlth Sci Univ L334,3181 SW SamJackson Pk Rd Portland OR USA 97201
Citazione:
X.H. Liu et al., "CFTR: Covalent modification of cysteine-substituted channels expressed in Xenopus oocytes shows that activation is due to the opening of channels resident in the plasma membrane", J GEN PHYSL, 118(4), 2001, pp. 433-446

Abstract

Sonic studies of CFTR imply that channel activation can be explained by anincrease in open probability (P-o), whereas others suggest that activationinvolves an increase in the number of CFTR channels (N) in the plasma membrane. Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substitutedconstruct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+). Covalent modification of R334C CFTR with MTSET+ doubled the conductance and changed the IN relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Thus, labeled and unlabeled channels could be differentiated by noting the percent decrease in conductance brought about by exposure to 2-ME. When oocytes were briefly (20 s) exposed to MTSET+ before CFTR activation, the subsequently activatedconductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSET+. The addition Of unlabeled, newly synthesized channels to the plasma membrane could be monitored on-line during the time when the rate of addition was most rapid after cRNA injection. The addition of new channels could be detected as early as 5 It after cRNA injection, occurred with a half time of similar to 24-48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. These findings demonstrate that in Xenopus oocytes, the major mechanism of CFTR activation by cAMP is by means of an increase in the open probability of CFTR channels.

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Documento generato il 19/09/20 alle ore 21:29:42