Abstract
Previously, we observed that the cystic fibrosis transmembrane conductance regulator (CFTR) channel openings are destabilized by replacing several acidic residues in the amino-terminal tail with alanines (Naren, A. P., Cormet-Boyaka, E., Fu, J., Villain, M., Blalock, J. E., Quick, M. W., and Kirk, K. L. (1999) Science 286, 544-548). Here we determined whether this effect is due to the loss of negative charge at these sites and whether the amino-terminal tail also modulates other aspects of channel gating. We introduced cysteines at two of these positions (E54C/D58C) and tested a series of methanethiosulfonate (MTS) reagents for their effects on the gating properties of these cysteine mutants in intact Xenopus oocytes and excised membrane patches. Covalent modification of these sites with either neutral (MMTS) or charged (2-carboxyethylmethanethiosulfonate (MTSCE) and 2-(trimethylammonium)ethylmethanethiosulfonate (MTSET)) reagents markedly inhibited channel open probability primarily by reducing the rate of channel opening. The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions. The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could be reversed by the reducing agent dithiothreitol (200 microm) or by elevating the bath ATP concentration above that required to activate maximally the wild type channel (>1 mm). Interestingly, the three MTS reagents had qualitatively different effects on the duration of channel openings (i.e. channel closing rate), namely the duration of openings was negligibly changed by the neutral MMTS, decreased by the positively charged MTSET, and increased by the negatively charged MTSCE. Our results indicate that the CFTR amino tail modulates both the rates of channel opening and channel closing and that the negative charges at residues 54 and 58 are important for controlling the duration of channel openings.
Highlights
We observed previously [13, 14] that a cluster of negatively charged residues in a putative helical region of the aminoterminal tail (N-tail) participates in CFTR channel gating
Our results indicate that modification of these cysteines with either uncharged or charged MTS reagents markedly and reversibly reduces channel opening rate in excised membrane patches
Our results indicate that the amino-terminal tail can modulate both the opening and closing of the CFTR channel and that the duration of channel openings is dependent on the negative charges at positions Glu-54 and Asp-58
Summary
Mutagenesis—The E54C and E54C/D58C CFTR mutants were generated by PCR mutagenesis. A 1-kilobase pair fragment of wild type CFTR in pCDNA3 (Invitrogen Corp.) was amplified by PCR in the presence of an appropriate mutagenic oligonucleotide. Wild type and mutant CFTR cRNAs were injected into oocytes 2–5 days before voltage clamp studies or patch clamp recording. Two microelectrode voltage clamp experiments were performed to measure the macroscopic currents mediated by wild type CFTR and the cysteine mutants in intact oocytes. For patch clamp experiments the oocytes were shrunken briefly in a bath solution containing 140 mM N-methyl-D-glucamine, 0.5 MgCl2, 1 mM EGTA, 10 mM HEPES (pH 7.4 with HCl). Absolute values of opening rates and Po can be overestimated for patches containing channels with low activity for which the numbers of channels may be underestimated This potential source of error does not affect estimates of the relative changes of these parameters in paired experiments performed on individual excised patches.
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