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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.

Liu X, Smith SS, Sun F, Dawson DC - J. Gen. Physiol. (2001)

Bottom Line: Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+).The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of approximately 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.

View Article: PubMed Central - PubMed

Affiliation: Oregon Health Sciences University, Portland, OR 97201, USA.

ABSTRACT
Some studies of CFTR imply that channel activation can be explained by an increase in open probability (P(o)), whereas others suggest that activation involves 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-substituted construct (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 I-V 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 activated conductance 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 h after cRNA injection, occurred with a half time of approximately 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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I-V relationship of R334C CFTR is modified by MTSET+ and MTSES−. (A) I-V plots at steady-state activation. Oocytes were continuously perfused with a cocktail containing 10 μM isoproterenol and 1 mM IBMX (control). An ∼5-min exposure to 1 mM MTSET+ induced an approximate doubling of the conductance and a change in the shape of the I-V plot. (B) I-V plots obtained at steady-state activation (control) and after ∼5-min exposure to 1 mM MTSES− that attenuated the conductance by ∼50% and enhanced inward rectification.
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Figure 1: I-V relationship of R334C CFTR is modified by MTSET+ and MTSES−. (A) I-V plots at steady-state activation. Oocytes were continuously perfused with a cocktail containing 10 μM isoproterenol and 1 mM IBMX (control). An ∼5-min exposure to 1 mM MTSET+ induced an approximate doubling of the conductance and a change in the shape of the I-V plot. (B) I-V plots obtained at steady-state activation (control) and after ∼5-min exposure to 1 mM MTSES− that attenuated the conductance by ∼50% and enhanced inward rectification.

Mentions: The effects of MTSET+ and MTSES− modification on the conductance of R334C CFTR are documented in the companion paper (see Smith et al. 2001, in this issue) and were briefly summarized in Fig. 1. Expression of R334C CFTR in Xenopus oocytes gives rise to cAMP-activated Cl− conductance characterized by modest inward rectification, which is distinct from that seen with expression of wt CFTR that is characterized by modest outward rectification. Brief exposure of oocytes expressing R334C CFTR to MTSET+ results in an approximate doubling of the conductance and a change in the shape of the I-V plot to one that is linear. In contrast, application of MTSES− attenuates the conductance by ∼50% and enhances the inward rectification. Recordings from excised patches presented in the companion paper (see Smith et al. 2001, in this issue) also demonstrated that MTSET+ modification increased the single-channel conductance of R334C CFTR. The effect of MTSET+-modification was not spontaneously reversible, but was readily reversed by a reducing reagent such as 2-ME (see Fig. 3). These observations indicated that MTSET+-modified and -unmodified channels could be distinguished by their functional characteristics.


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.

Liu X, Smith SS, Sun F, Dawson DC - J. Gen. Physiol. (2001)

I-V relationship of R334C CFTR is modified by MTSET+ and MTSES−. (A) I-V plots at steady-state activation. Oocytes were continuously perfused with a cocktail containing 10 μM isoproterenol and 1 mM IBMX (control). An ∼5-min exposure to 1 mM MTSET+ induced an approximate doubling of the conductance and a change in the shape of the I-V plot. (B) I-V plots obtained at steady-state activation (control) and after ∼5-min exposure to 1 mM MTSES− that attenuated the conductance by ∼50% and enhanced inward rectification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233697&req=5

Figure 1: I-V relationship of R334C CFTR is modified by MTSET+ and MTSES−. (A) I-V plots at steady-state activation. Oocytes were continuously perfused with a cocktail containing 10 μM isoproterenol and 1 mM IBMX (control). An ∼5-min exposure to 1 mM MTSET+ induced an approximate doubling of the conductance and a change in the shape of the I-V plot. (B) I-V plots obtained at steady-state activation (control) and after ∼5-min exposure to 1 mM MTSES− that attenuated the conductance by ∼50% and enhanced inward rectification.
Mentions: The effects of MTSET+ and MTSES− modification on the conductance of R334C CFTR are documented in the companion paper (see Smith et al. 2001, in this issue) and were briefly summarized in Fig. 1. Expression of R334C CFTR in Xenopus oocytes gives rise to cAMP-activated Cl− conductance characterized by modest inward rectification, which is distinct from that seen with expression of wt CFTR that is characterized by modest outward rectification. Brief exposure of oocytes expressing R334C CFTR to MTSET+ results in an approximate doubling of the conductance and a change in the shape of the I-V plot to one that is linear. In contrast, application of MTSES− attenuates the conductance by ∼50% and enhances the inward rectification. Recordings from excised patches presented in the companion paper (see Smith et al. 2001, in this issue) also demonstrated that MTSET+ modification increased the single-channel conductance of R334C CFTR. The effect of MTSET+-modification was not spontaneously reversible, but was readily reversed by a reducing reagent such as 2-ME (see Fig. 3). These observations indicated that MTSET+-modified and -unmodified channels could be distinguished by their functional characteristics.

Bottom Line: Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+).The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of approximately 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.

View Article: PubMed Central - PubMed

Affiliation: Oregon Health Sciences University, Portland, OR 97201, USA.

ABSTRACT
Some studies of CFTR imply that channel activation can be explained by an increase in open probability (P(o)), whereas others suggest that activation involves 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-substituted construct (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 I-V 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 activated conductance 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 h after cRNA injection, occurred with a half time of approximately 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.

Show MeSH
Related in: MedlinePlus