©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternate Translation Initiation Codons Can Create Functional Forms of Cystic Fibrosis Transmembrane Conductance Regulator (*)

Tiziana Piazza Carroll , Marcelo M. Morales , Stephanie B. Fulmer , Sandra S. Allen , Terence R. Flotte , Garry R. Cutting , William B. Guggino (§)

From the (1) Department of Physiology and Pediatrics and the Center for Medical Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
REFERENCES

ABSTRACT

To evaluate the function of transmembrane domain 1 (TMD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and the methionines that function in translation initiation, a series of progressive 5` truncations in TMD1 were created to coincide with residues that might serve as translation initiation codons. Expression of the mutants in Xenopus oocytes demonstrated that internal sites in TMD1 can function as initiation codons. In addition, all of the mutants that progressively removed the first four transmembrane segments (M1-M4) of TMD1 expressed functional cAMP-regulated Cl channels with ion selectivity identical to wild-type CFTR but with reduced open probability and single channel conductance. Further removal of transmembrane segments did not produce functional Cl channels. These data suggest that segments M1-M4 are not essential components of the conduction pore or the selectivity filter of CFTR.


INTRODUCTION

Cystic fibrosis is a lethal genetic disease caused by mutations of CFTR()(1, 2, 3, 4, 5, 6, 7) . CFTR is composed of five domains: two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and one regulatory domain (1) . CFTR functions both as a secretory Cl channel (8, 9, 10, 11) and as a conductance regulator (12) . Mutations causing severe disease either dramatically alter channel function as with G551D or affect channel trafficking to the plasma membrane as with F508 (13, 14, 15, 16) . In contrast, the characteristics of the mild mutations in TMD1 are quite similar to wild-type CFTR including ion selectivity, cAMP-dependent protein kinase A phosphorylation, and ATP dependence (17, 18, 19) .

TMD1 is thought to play a role in formation of the channel pore (10, 20, 21, 22) . Anderson et al.(10) demonstrated that mutation of lysine 95 or 335 alters CFTR's anion selectivity and concluded that these amino acids play a key role in the selectivity filter. Akabas et al.(21) demonstrated that cysteines substituted for Gly, Lys and Gln in the M1 domain (M represents individual membrane spanning domain) are accessible to cysteine reactive reagents and concluded that these residues are in the pore. In contrast Oblatt-Montal et al. (20) showed that peptides with sequences corresponding to M1, M3, M4, and M5 do not form channels, whereas only hetero-oligomers of M2 and M6 exhibit channel characteristics that emulate wild-type CFTR. McDonough et al.(22) also concluded that M6 plays a critical role in conduction. They, in addition, implicated M12 as a component of the pore structure of CFTR and hypothesized that both M6 and M12 interact to form the pore. We have shown (12) that CFTR missing the first 119 amino acids in which M1 has been completely removed and partially replaced with 22 amino acids with no homology to CFTR has single channel characteristics very similar to wild type (12) . Because M1 includes Gly, Lys, Gln, and Arg this observation suggests that these residues are not essential for either channel conduction or selectivity. Taken together, it is clear that a complete picture of which amino acids actually line the pore is still lacking.

It is known that the 5` end of CFTR mRNA transcripts can be alternately spliced in epithelial tissues. The resultant transcripts are missing exon 1, which contains the canonical translation initiation codon (23) . We demonstrated in this study that internal sites can function as translation initiation codons and form functional, cAMP-regulated Cl channels.


MATERIALS AND METHODS

Construction of Mutants

CFTR constructs are illustrated in Fig. 1. Point mutations were created in CFTR cDNA clone pBQ4.7 by oligonucleotide-mediated, single strand mutagenesis using the Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad) (24) . Missense mutations were introduced using the following primers: M1V, 5`-ACC CCA GCG CTC GAG AGA CCG TGC AGA GGT-3; M150V, 5`-CAC ATT GGA GTG CAG ATG AG-3`; and 259-M265V, 5`-GAC TAG TGA TTA CCT CAG AAG TGA TTG-3`. A truncation at the 5` end of the CFTR cDNA, 259, was created by introducing a unique SpeI restriction site and digesting with that enzyme to remove the 5` portion of CFTR and religating the plasmid. 119, a deletion of the first 119 amino acids, was created by removing the Nru I/SmaI fragment of the CFTR cDNA and then religating the plasmid. All mutations are confirmed by DNA sequencing. Two additional CFTR variants, pSA306 and -S118 M (pTRF42) have been described previously (12, 25, 26) . pTRF42 was constructed by polymerase chain reaction modification to contain a unique initiation codon directly upstream from Ile of CFTR. The CFTR-containing open reading frame of each of these constructs was cloned into pBluescript SK+ for in vitro transcription. All the other mutants in pBQ4.7 were transcribed in vitro using a Megascript kit (Ambion). cRNA was injected into Xenopus oocytes for assay of CFTR Cl function.


Figure 1: Mutations in TMD1. A schematic representation of WT and mutant forms of CFTR is shown. The representation is based on the putative model for WT CFTR (1). Each smallrectangle represents a transmembrane domain segment, while straightlines are the intra- or extracytosolic loops. Biggerrectangles are NBDs and the oval is the regulatory domain. The flatwhiterectangle at the beginning of pSA306 represents the 26-amino acid ``flag'' inserted in this sequence. Methionines at sites that fit the consensus for translation initiation (27) are indicated by an arrow.



Assay of CFTR in Xenopus Oocytes

Oocytes were prepared as described previously (19) . Two microelectrode voltage clamp measurements were carried out 72 h following injection of mRNA or water. Oocytes injected with mutant and wild-type CFTR were prestimulated with forskolin (10 uM) and IBMX (1 mM). This mixture is well known to activate CFTR expressed in oocytes (19) . cAMP-activated currents are referred to as I. The voltage step protocol involved changing the potential from -90 to +50 at 20-mV step intervals of 600 ms. The potential was returned to the holding potential (-70 mV) for 600 ms between each step. Oocytes were injected with 50 nl of either water experiments or a solution containing 0.1-1 µg/µl RNA. The bath solution contained (in mM) 115 NaCl, 2 KCl, 1 CaCl, 1 MgCl, and 5 Hepes adjusted to pH 7.4 with NaOH. One or more wild type- and water-injected oocytes were assayed before mutant forms of CFTR were assessed. When no currents were generated by wild-type CFTR, the whole experiment was abandoned. I were never observed in water injected oocytes. When the Cl was reduced to evaluate the reversal potential, base-line currents prior to stimulation were subtracted from forskolin and IBMX-activated values.

For patch clamping, oocytes were placed in hypertonic solution (475 mosm) containing (in mM) 200 potassium aspartate, 20 KCl, 1 MgCl, 10 EGTA, 10 Hepes at pH. 7.4 (see Ref. 19). The vitelline membrane was removed with forceps. Oocytes were then transferred back into oocyte Ringer solution. The same solution was also used in the patch pipette. Oocytes were prestimulated with forskolin (10 µM) and IBMX (1 mM) to activate CFTR. Patches were excised in the presence of protein kinase A (50 nM) and MgATP (1 mM) to prevent rundown. To measure ion selectivity bath Cl was replaced by either I or Br. Data recording, analysis, and patch clamp equipment were identical to those already published (12, 19) .

RESULTS

Deletion of Transmembrane Segment 1

To test whether CFTR missing the M1 segment can function as a Cl selective ion channel, two different M1 deletion mutants were created and analyzed in Xenopus oocytes (Fig. 1). pSA306 CFTR has the first 118 amino acids of CFTR replaced with a 26-amino acid epitope tag that has no homology with the original sequence (26) . -S118M is a mutant with the first 117 amino acids of CFTR removed and a substitution of a methionine at position 118. I from oocytes injected with either of the mutants (Fig. 2A) had current versus voltage relationships (I/V) with reversal potentials consistent with Cl currents (Fig. 2B). As much as 500 µM DIDS was without effect (n = 3 experiments for each of the mutants, data not shown). When the Cl concentration in the bath was lowered from 121 to 30 mM the reversal potential shifted to positive values as expected for Cl currents (pSA306: -25.5 ± 0.6 to +5.4 mV ± 0.8, n = 3, and -S118M: -26.2 ± 0.8 to +4.46 mV ± 0.6, n = 3). These data verify previous data (19) indicating that the expressed currents are indeed generated specifically by CFTR.


Figure 2: Wild-type CFTR, pSA306, and -S118M. A, representative whole-cell current recordings of WT CFTR (a and b), pSA306 (c and d), and -S118M (e and f). a, c, and e are the base-line currents measured in the oocytes prior to stimulation for each respective mutation. The voltage protocol is depicted in the upperrightcorner. B, representative I/V curve calculated from the data in A. The outwardrectification of the curves is due to the higher extracellular concentration of chloride (121 mM) compared with the inside of the oocyte (40 mM). Several experiments were performed. Average whole currents measured at +50 mV are as follows: WT, 1799 ± 105 nA (n = 5); pSA306, 1244 ± 72 nA (n = 5); -S118M, 1329 ± 61 nA (n = 5). C, representative single channel recordings from inside-out patches. The oocytes were injected with WT CFTR (a), pSA306 (b), or -S118M (c) DNA. Voltages were +53 mV for a, +51 mV for b, and +51 mV for c. Arrows indicates close channel state.



To characterize in more detail the function of the two mutants, excised single channel patch clamp experiments were performed. Tracings from CFTR-injected oocytes are shown in Fig. 2C. CFTR-like channel activity was not observed in water-injected oocytes under similar conditions. Single channel conductance and open probability of both mutants were smaller than WT CFTR but very similar to each other (). Anion permeability of the mutants was identical to those of WT CFTR (Br > Cl > I). The relative anion conductance Br/Cl for pSA306 was 1.35 ± 0.07 (n = 3), and for -S118M it was 1.37 ± 0.06 (n = 3) at 50 mV. Channel conductances for both WT and mutant forms of CFTR in iodide-containing solutions were too small to measure. Thus channels either completely missing M1 or possessing a small 26-amino acid ``epitope tag'' still form Cl channels, with characteristics very similar to WT CFTR.

Alternate Translation Initiation

To address whether sites within WT CFTR can function as initiation codons, a series of mutants was made including M1V (substitution of the first methionine with valine), 119 (removal of CFTR sequences prior to 120 with the next methionine in a suitable context for translation initiation at Met), and M1V-M150V (substitution of both methionines at 1 and 150 with valine). All of these mutants generated chlorine currents (Fig. 3A) very similar to WT CFTR. All of the currents from the mutants were insensitive to DIDS and time-independent. The I/V relationship was similar to WT. The reversal potential for each of the mutants is consistent with Cl currents (Fig. 3B). When the Cl concentration in the bath was lowered from 121 to 30 mM the reversal potential shifted to positive values as expected for Cl currents (M1V: -23.8 ± 1.5 to +4.7 ± 0.06 mV, n = 2; 119: -22.7 ± 1.2 to +5.3 ± 0.8 mV, n = 2; M1V-M150V: -24.4 ± 1.2 to +5.9 ± 0.4 mV, n = 2). Again, no CFTR-like Cl currents were observed either in mutant- or wild type- injected oocytes prior to activation with forskolin and IBMX. These results demonstrate that the first methionine is not unique for initiation of translation of CFTR and that downstream codons can also function as translation initiation sites. Generation of Cl currents by the 119(M150), which does not contain any CFTR sequence prior to amino acid 120 indicates that the next likely translation initiation site (27) is at M150. Expression of Cl currents from the double mutant, M1V-M150V, suggests that methionines beyond 150 may also initiate translation in Xenopus oocytes.


Figure 3: WT CFTR, M1V, 119, and M1V-M150V. A, representative whole-cell current recordings from WT CFTR (a and b), M1V (c and d), 119 (e and f), and M1V-M150V (g and h). a, c, e, and g are the base-line currents measured in the oocytes prior to stimulation for each respective mutation. The voltage protocol is depicted in the upperleftcorner. B, I/V relationship of the representative data shown in A. The intercept of the curves with the x axis indicates the reversal potential for WT CFTR and the mutants. Several experiments were performed. Average whole currents measured at +50 mV were as follows: WT, 917 ± 136 nA (n = 2); M1V, 614 ± 113 nA (n = 6); 119, 606 ± 101 nA (n = 5), M1VM150V, 452 ± 92 nA (n = 4).



To verify that several sites in CFTR can function as Kozak initiation codons (27) , we made the following mutants, 259 and 259-M265V; both of these mutants have all of the coding sequence removed prior to amino acid 259. 259 has an excellent initiation site at M265, and generates I with overall characteristics very similar to WT CFTR: time independence (Fig. 4A), DIDS insensitivity (n = 5, data not shown), and a reversal potential expected for a Cl current (Fig. 4B). When Cl in the bath is lowered to 30.25 mM the reversal potential shifts to positive values (-20.2 ± 1.9 to 4.4 mV ± 0.88, n = 3) consistent with a chloride current. No currents were detected in injected oocytes prior to activation (Fig. 4A). These data suggest that the protein translated by CFTR mRNA missing methionines at positions 1 and 150 most likely uses M265 to initiate translation. 259-M265V mRNA was injected in oocytes, but no forskolin and IBMX-generated currents were detected (n = 6). This suggests either that the 259-M265V mutant is not translated even though there is methionine at position 281, which is in a suitable context for translation initiation, that the mutant is not processed to the plasma membrane, or that the mutant, 259 CFTR, included the smallest amount of TMD1 that can form functional Cl channels.


Figure 4: Wild-type CFTR and 259. A, representative whole-cell currents for WT CFTR (a and b) and 259 (c and d). a and c are the unstimulated base-line currents for WT and 259, respectively; voltage protocol is depicted in the leftportion of the picturecenter. B, I/V relationship of the data in A. Several experiments were performed. Average whole currents measured at +50 mV were as follows: WT, 2218 ± 542, (n = 4); 259 = 1106 ± 291 nA (n = 11). C, representative single channel recordings from inside-out patches of oocytes injected with 259 mRNA. The arrow indicates close channel state. Voltage is +59 mV.



To characterize 259, the single channel properties were compared with WT CFTR. Fig. 4C illustrates a current tracing recorded at +55 mV. The conductance and the open probability are smaller than WT CFTR (). Despite the removal of the first four transmembrane segments of CFTR M1-4, the anion selectivity of 259 is not different from WT CFTR (Br > Cl > I). The ratio of bromide to chloride conductance is 1.36 ± 0.12 (n = 3) for 259 and 1.4 ± 0.08 (n = 3) for WT. Channel conductances for both WT CFTR and 259 in iodine-containing solutions were too small to measure.

DISCUSSION

We have shown that removal of a large portion of TMD1 including segments M1-M4 does not alter the ion selectivity of CFTR. Further removal of portions of CFTR beyond M4 produces a nonfunctional protein either because the protein is not translated or processed properly or because a critical component of the channel pore is located in M5 and M6. Evidence that M5 and M6 may line the channel pore was provided by Tabcharani et al.(28) . They demonstrated that CFTR has a multi-ion pore and that mutating Arg in M6 alters both the conductance and converts CFTR to a single-ion pore. Arg is also the site of several naturally occurring mutations that are associated with mild airway disease (5, 29) . Thus, these mutations may compromise CFTR function by affecting the channel pore directly.

Because all deletions of M1-M4 reduce the single channel conductance of CFTR only modestly (the maximum effect is a 30% reduction when all four domains are removed) without affecting ion selectivity, it is possible that segments M1-M4 line distant portions of the pore. Interestingly, the reduction in conductance with the TMD1 deletion mutants is similar to that observed with the naturally occurring mild mutation R117H, which results in an approximate 25% reduction in conductance (19) . However, the observation that both truncation mutants and R117H mutant (17, 19) affect conduction without any changes in selectivity demonstrate clearly that the M1-M4 segments are not critical for maintaining channel selectivity. Prior experiments have shown that mutating a lysine residue at position 95 to an acidic amino acid alters the selectivity sequence of the channel to I > Br > Cl, suggesting that Lys plays a role in determining anion selectivity. Why does K95D alter selectivity whereas removal of all of M1 does not? One explanation is that this mutant is not directly involved in the selectivity filter but affects selectivity via an allosteric effect on another domain of CFTR. Changing the charge at position 95 from a positively charged lysine to a negatively charged aspartate may affect the folding of the entire transmembrane domain, which would be expected to affect the selectivity filter indirectly. This could occur if the charge Lys in wild-type CFTR is normally paired with a negatively charged residue such as Glu. If the positively charged Lys is indeed paired with a negatively charged amino acid such as glutamate, then mutating Lys to aspartate would create a highly polar region within a transmembrane domain that may destabilize TMD1. Removal of the entire M1 segment may not be expected to have such a profound effect.

CFTR activation is a two-step process requiring both the phosphorylation by protein kinase A and the binding of ATP to NBDs. A hypothesis regarding the nature of CFTR gating has emerged recently, that the degree of phosphorylation of CFTR modulates an interaction between the two NBDs (30) . The interaction is such that one NBD controls channel opening and the other closing. Relevant to this study is that single amino acid substitutions such as R117H and truncations such as removal of M1-M4 of TMD1 reduce the channel open probability by about 30-40% when activated under identical conditions. With the mutant such as R117H, which occurs in the first putative exofacial loop of CFTR, and with all of the truncation mutants a reduction in open probability could suggest that portions of segments M1-M4 may play a minor role in channel gating but clearly are not critical for activation of CFTR by protein kinase A and ATP.

Several sites in CFTR can potentially function as translation initiation codons in Xenopus oocytes. It is known that CFTR has alternative exons, -1a and 1a, located upstream of exon 1 (23) . RNA studies have identified in T84 and CACO-2 epithelial cell lines two transcripts containing these exons, one in which exon -1a is spliced to 1a and then to exon 2 and the other in which -1a is spliced directly to exon 2. Neither of these transcripts contains AUG initiation codons in the correct reading frame requiring the use either of unconventional start codons (i.e. CVG) or downstream AUG codons in exon 4. We demonstrate that alternate codons can function as sites for translation initiation and that the truncated CFTR isoforms produce an ion channel with the same anion selectivity, as wild type but with a somewhat reduced single channel conductance and open probability. If these alternately spliced forms of CFTR are expressed and translated in human cells, they could contribute significantly to overall Cl secretion in vivo.

  
Table: WT CFTR, pSA306, -S118M, and 259 single channel characteristics

Data are mean ± S.E. n = number of active patches.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL47122, HL51811, DK48977, and DK44003 and by the Cystic Fibrosis Foundation Research Development Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology and Pediatrics, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-7166 or 410-955-4816; Fax: 410-955-0461; E-mail: wguggin@wpo.bs.jhu.edu.

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; TMD, transmembrane domain; NBD, nucleotide binding domain; IBMX, isobutylmethylxanthine; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid; WT, wild type.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.