ABCMdb

PMID: 11585853

Liu X, Smith SS, Sun F, Dawson DC
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 Physiol. 2001 Oct;118(4):433-46., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Arg334Cys 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 (MTSETϩ). Login to comment 3 ABCC7 p.Arg334Cys Covalent modification of R334C CFTR with MTSETϩ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Login to comment 5 ABCC7 p.Arg334Cys When oocytes were briefly (20 s) exposed to MTSETϩ 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 MTSETϩ. Login to comment 7 ABCC7 p.Arg334Cys The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of ~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. Login to comment 18 ABCC7 p.Arg334Cys One of these cysteine-substituted constructs (R334C) was readily modified by MTS reagents in the external bath and covalent modification gave rise to the changes in anion conduction that could be easily detected in the macroscopic I-V plots recorded in Xenopus oocytes, permitting us to distinguish modified from unmodified-channels. Login to comment 19 ABCC7 p.Arg334Cys R334C CFTR, in conjunction with membrane impermeant thiol reagent, MTSETϩ (Holmgren et al., 1996), offered an opportunity to test directly the hypothesis that activation of Cl-conductance in oocytes expressing CFTR is accompanied by an increase in channel number in the plasma membrane. Login to comment 20 ABCC7 p.Arg334Cys We found that exposure of oocytes expressing R334C CFTR to MTSETϩ for 20 s before activation resulted in labeling of the entire membrane pool of functional channels, suggesting that channels activated by cAMP are resident in the membrane before activation and that activation of Cl-conductance, therefore, is due largely to an increase in the open probability (Po) of CFTR channels. Login to comment 55 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys R E S U L T S Modification of R334C CFTR by MTSETϩ Is Stable but Reversible The effects of MTSETϩ 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. Login to comment 56 ABCC7 p.Arg334Cys 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. Login to comment 57 ABCC7 p.Arg334Cys Brief exposure of oocytes expressing R334C CFTR to MTSETϩ results in an approximate doubling of the conductance and a change in the shape of the I-V plot to one that is linear. Login to comment 59 ABCC7 p.Arg334Cys Recordings from excised patches presented in the companion paper (see Smith et al., 2001, in this issue) also demonstrated that MTSETϩ modification increased the single-channel conductance of R334C CFTR. Login to comment 69 ABCC7 p.Arg334Cys I-V relationship of R334C CFTR is modified by MTSET؉ and MTSES-. Login to comment 75 ABCC7 p.Arg334Cys Modification of R334C CFTR by MTSET؉ was stable for at least 5 h. Login to comment 80 ABCC7 p.Arg334Cys also characteristic of R334C CFTR as described in the accompanying paper (see Smith et al., 2001, in this issue) and Fig. 1 and did not change with time. Login to comment 81 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys The Entire Membrane Pool of R334C CFTR Activated by cAMP Is Accessible to MTSETϩ before Activation The level of CFTR expression used in these studies was such that, before activation by stimulatory cocktail, the conductance of the oocyte membrane was similar to that seen in an oocyte that was not expressing R334C CFTR. Login to comment 84 ABCC7 p.Arg334Cys Fig. 3 contains plots of the conductance (@Erev) versus time obtained from a group of experiments designed to determine if R334C CFTR channel can be modified by externally applied, impermeant thiol reagents in the active as well as the inactive state. Login to comment 87 ABCC7 p.Arg334Cys The efficacy of MTSETϩ modification of R334C CFTR did not depend on the state of activation of CFTR (Fig. 3 B). Login to comment 91 ABCC7 p.Arg334Cys The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with MTSET؉ before the activation. Login to comment 92 ABCC7 p.Arg334Cys Records of gCl versus time obtained from R334C CFTR expressing oocytes that were obtained from the same frog and assayed on the same day. Login to comment 98 ABCC7 p.Arg334Cys of the oocyte to MTSETϩ and 2-ME induced the same response, suggesting that pre-and postactivation exposure to MTSETϩ labeled the same population of R334C CFTR channels. Login to comment 99 ABCC7 p.Arg334Cys To determine if labeling of the entire pool of R334C CFTR channels could be attributed to nonreacted MTSETϩ that might remain after washing and, thus, be present during channel activation, the channels were Figure 4. Login to comment 100 ABCC7 p.Arg334Cys MTSET؉ labeling did not affect the activation and inactivation process of R334C CFTR. Login to comment 106 ABCC7 p.Arg334Cys The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with 20-s exposure to MTSET؉ before the activation. Login to comment 115 ABCC7 p.Arg334Cys To determine if labeling with MTSETϩ altered the process of R334C CFTR activation, we first labeled the channels with 100 ␮M MTSETϩ after activation, then inactivated the labeled channels, and then reactivated them at a later time (Fig. 4). Login to comment 118 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Reactivation of R334C CFTR increased gCl to a level similar to that seen after the first modification, and 2-ME decreased gCl by ‫.%05ف‬ The effects were reproduced by the second exposure to MTSETϩ and 2-ME, indicating that MTSETϩ did not interfere with the activation or inactivation of R334C CFTR. Login to comment 124 ABCC7 p.Arg334Cys The shape of the I-V plot obtained at steady-state activation in the presence of MTSES- was characteristic of MTSETϩ- modified R334C CFTR (Fig. 5 C, 3), indicating that prelabeling with MTSETϩ for 20 s prevented modification by MTSES- before and during activation. Login to comment 126 ABCC7 p.Arg334Cys Exposure of the oocyte to the reducing reagent (2-ME) decreased the conductance to about half the stimulated value, and changed the shape of the I-V plot to inward rectification typical of unmodified R334C (Fig. 5 C, 6), indicating that the positively charged, TEA group that was added in the inactive state was readily removed. Login to comment 128 ABCC7 p.Arg334Cys The results indicate that the modification of R334C CFTR by MTSETϩ was complete, regardless of whether exposure to the reagent took place in the active or inactive state, confirming that the entire pool of CFTR channels was accessible to MTSETϩ before activation. Login to comment 129 ABCC7 p.Arg334Cys The Time Course of Addition of R334C CFTR Channels to the Plasma Membrane after cRNA Injection After cRNA injection, new CFTR channels must be synthesized and inserted in the oocyte plasma membrane. Login to comment 131 ABCC7 p.Arg334Cys Time-dependent increase in the conductance of R334C CFTR in the first 5 d after cRNA injection. Login to comment 138 ABCC7 p.Arg334Cys Fig. 6 is a summary of the conductance of R334C CFTR in its inactive and active state during the first 5 d after cRNA injection. Login to comment 171 ABCC7 p.Arg334Cys The oocytes used in this group of experiments were obtained from the same frog and were injected with R334C CFTR RNA at the same time. Login to comment 174 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys D I S C U S S I O N In CFTR-expressing Oocytes cAMP Increases Cl-Conductance by Increasing the Open Probability of Channels The results presented here are consistent with the notion that the entire, activatable pool of R334C CFTR in the Xenopus oocyte is accessible to MTSETϩ during a 20-s exposure to a perfusion solution containing the reagent. Login to comment 187 ABCC7 p.Arg334Cys MTSETϩ Is Impermeant The studies of Holmgren et al. (1996) and Yang et al. (1996) suggested that MTSETϩ is not likely to cross the plasma membrane and label R334C CFTR protein that might exist in subplasma membrane vesicles. Login to comment 209 ABCC7 p.Arg334Cys If endocytosis of R334C CFTR were proceeding in Xenopus oocytes at the highest rate reported for mammalian cells (‫/%05ف‬min), then the maintenance of the steady-state, activated CFTR conductance commonly observed in oocytes would require an equally high exocytotic delivery rate to maintain the channel population. Login to comment 211 ABCC7 p.Arg334Cys This sort of behavior was never observed, suggesting that the rate of turnover of R334C CFTR is relatively slow in Xenopus oocytes. Login to comment 218 ABCC7 p.Arg334Cys It must also be emphasized that all of the results reported here were obtained using a CFTR mutant (R334C), so that it is possible this mutation suppresses stimulation-dependent trafficking mechanism that is more prominent in the wild-type and M2-901 CFTR (see Mechanism of CFTR Activation). Login to comment 219 ABCC7 p.Arg334Cys However, like other membrane proteins that undergo clathrin-mediated endocytosis, the internalization signal contained in the cytoplasmic tail of the wt CFTR (which was retained in R334C CFTR) was found to be sufficient for promoting endocytosis (Prince et al., 1999). Login to comment 220 ABCC7 p.Arg334Cys It also seems unlikely that labeling of R334C CFTR with thiol reagents would cause the apparent low rate of recycling, given that biotinylated CFTR is efficiently endocytosed in mammalian cells (Prince et al., 1994; Lukacs et al., 1997). Login to comment 221 ABCC7 p.Arg334Cys Blocking the Insertion of Channels Did Not Affect Activation After labeling with MTSETϩ, the appearance of unlabeled channels on the plasma membrane was observed during the first few days after the injection of R334C cRNA when the insertion of new channels via the biosynthetic pathway was expected. Login to comment