From the
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
Cystic fibrosis is a lethal genetic disease caused by mutations
of
CFTR
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
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
For patch clamping, oocytes
were placed in hypertonic solution (475 mosm) containing (in
mM) 200 potassium aspartate, 20 KCl, 1 MgCl
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
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
Data are mean ± S.E. n = number of active
patches.
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.
(
)(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) .
, 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.
channels.
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.
, 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.
> 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.
secretion in vivo.
Table:
WT CFTR,
pSA306, -S118M, and
259 single channel characteristics
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.