(Received for publication, October 3, 1996, and in revised form, December 15, 1996)
From the Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York, New York 10029
Heterologous coexpression of recombinant, G protein-gated, inwardly rectifying K+ (GIRK) channel subunits has yielded large currents, severalfold greater than those obtained from expression of the individual subunits. Such current enhancement has been obtained from coexpression of the inactive GIRK1 subunit with the low activity GIRK2-5 subunits in Xenopus oocytes. Using deletion and chimeric constructs, we now report the identification of a C-terminal region unique to GIRK1 and a larger central region of GIRK4 highly homologous to GIRK1, both of which are critical for production of large currents. Chimeras containing these two regions produced homomeric channels, exhibiting currents severalfold greater than those from either wild-type subunit alone. G protein regulation of such chimeric channel currents resembled that of wild-type currents. Green fluorescent protein-tagged channels showed that the amount of chimeric channel expressed on the oocyte cell surface was similar to its wild-type counterpart, suggesting that the enhanced activity was not due to differences in relative levels of expression but rather to the coexistence of the chimeric regions. Single-channel recordings of the active chimeras exhibited patterns of activities with open-time kinetics and conductance characteristics representative of those of GIRK4, indicating that the presence of the GIRK1 C-terminal region caused an increase in the frequency of channel openings without affecting their duration.
G protein-gated inwardly rectifying K+
(GIRK)1 channels use a membrane delimited
mechanism to provide cardiac, neuronal, and endocrine cells with rapid
inhibition of membrane excitability in response to extracellular
signals. The best studied case, the atrial ACh-induced K+
channel, has been shown to be directly activated by the subunits of G proteins in response to the binding of acetylcholine to the muscarinic m2 receptor (1). Similar GIRK channels have also been found
in neuronal tissues (2). Five homologous members, GIRK1 through GIRK5,
have been identified in this channel subfamily (3-7). Heterologous
expression of individual GIRK members has produced relatively small
currents with short-lived single-channel events (6). Recent studies
from coimmunoprecipitation (6, 8) immunocytochemistry (9), in
situ hybridization (10-12), and functional coexpression (6-8,
13-17) have demonstrated the heteromeric nature of GIRK channels. The
GIRK1 subunit has been found to greatly enhance the activity and change
the single-channel kinetics of other GIRK members, even though when
expressed alone it fails to form functional homomeric channels (7).
Recently, we identified a single P-region residue of GIRK1,
Phe137, which when associated with other GIRK subunits
contributed to the current enhancement and prolonged the duration of
single-channel openings (17). However, additional determinants, other
than Phe137, were implicated for the high activity of
heteromeric channel currents (17).
We set out to identify such additional structural determinants of activity using the human GIRK1 and KGP (or GIRK4) channel subunits and monitoring their ability to cause current enhancement as a result of deletions and chimeric manipulations. In this study, we report that both the unique C terminus of GIRK1 and a homologous core region of KGP are involved in generation of enhanced currents. We have engineered chimeras containing these critical regions, which are capable of producing homomeric channels exhibiting large currents. These results revealed that specific regions of the two channel subunits are involved in producing large currents. Identification of these critical regions is an important step in elucidating how the two subunits interact to give rise to the highly active heteromeric channel.
GIRK1 and
KGP were each subcloned in pGEMHE and were used as template for PCR (8,
18). The K1K2G3 chimera (see Fig. 1 for nomenclature) was constructed using a three-fragment ligation reaction. The K1K2 fragment was amplified by
PCR with BamHI and NaeI sites incorporated in the
5 and 3
ends, respectively. Similarly, the G3 fragment
was amplified with DraI and EcoRI sites
incorporated at the ends. Both PCR fragments were subcloned into the
region between BamHI and EcoRI sites of pGEMHE to
create the full-length chimera. Restriction digestion with
SacI yielded a fragment spanning the junction between
K1K2 and G3 regions and expanding
through the stop codon (one SacI site was located within the
codons for amino acids 319-321 of KGP, whereas the other was in the
pGEMHE vector). Sequencing (19) of this SacI cassette
confirmed its integrity and revealed a single silent mutation
corresponding to Pro370 of GIRK1 (CCC was changed to CCT).
The SacI cassette was subcloned back into pGEMHE-KGP. A
similar three-fragment ligation strategy was used to construct the
G1G2K3 and
G1K2K3 chimeras.
G1K2G3 was constructed by splicing
G1K2K3 and
K1K2G3 using the common
KpnI site found in the K2 region. The integrity
of all chimeras was confirmed by sequencing.
K1G2G3 and K1K2I3 were constructed using a PCR splicing method (20). The full-length chimeras were subcloned into pGEMHE. The sequence of the full-length K1G2G3 was confirmed. As with K1K2G3, the sequence of a SacI cassette in the K1K2I3 chimera was verified after subcloning in pGEMHE-KGP.
GIRK1 deletion mutants were constructed by amplification of different
truncated versions of GIRK1 using T7 and different reverse primers. For
GIRK11 (Ser453-Thr501 were deleted) or
GIRK1
2 (Arg409-Thr501 were deleted), a
PstI cassette was sequenced and subcloned back into
pGEMHE-GIRK1. The same cassettes were subcloned to PstI-cut pGEMHE-K1K2G3 fragment to produce
K1K2G3
1 and
K1K2G3
2. Similarly, a
SphI-cut cassette of pGEMHE-G1G2
(Lys359-Thr501 were deleted) was sequenced and
subcloned back to pGEMHE-GIRK1. Similarly, a SacI-cut
cassette in the C terminus of pGEMHE-K1K2 was
sequenced and subcloned back to pGEMHE-KGP.
The stop codon was removed, and an EcoRV site was introduced
to the 3 end of both KGP and
K1K2G3 coding region using PCR, all
of which were confirmed by manual sequencing. The coding region of GFP
was amplified by PCR and subcloned in frame into EcoRV and
NotI sites to produce pGEMHE-KGP-GFP and
pGEMHE-K1K2G3-GFP.
All constructs were linearized with NheI, and cRNAs were transcribed as described previously (17). Oocytes were isolated and microinjected as described previously (21, 22). About 4 ng of each RNA species was injected per oocyte.
Two-electrode voltage clamp on Xenopus oocytes was performed as described previously (8, 23). The high potassium bath solution contained (in mM): 91 KCl, 1 NaCl, 1 MgCl2, 5 KOH/HEPES, 1.8 CaCl2, pH 7.4; high potassium with Ba2+ contained high potassium solution with 3 mM BaCl2. Each figure shows a representative experiment that includes oocytes from the same batch. A minimum of two to three batches of oocytes gave similar results.
Single-channel activity was recorded on devitellinized oocytes under
the cell-attached mode of standard patch-clamp methods (24, 25) using
an Axopatch 200A amplifier (Axon Instruments, CA) as described
previously (8). Single-channel recordings were performed at a holding
membrane potential of 80 mV in the absence of ACh from the high
potassium bath solution.
An antibody raised against the rat N-terminal 21 amino acids of CIR (Upstate Biotechnology, Lake Placid, NY) and an affinity purified C-terminal peptide antibody raised against the rat GIRK1 C-terminal end (Gln375-Thr501) (11) were used in immunoprecipitation experiments as described previously (8).
Evaluation of Surface Expression of Channel Proteins by Confocal MicroscopyXenopus oocytes were fixed in 4% paraformaldehyde at room temperature overnight. Fixed oocytes were embedded in 3% agarose, and 50-µm sections were cut, mounted, and viewed under a confocal microscope (Leica TCS 4D). In order to compare relative surface expression level of the KGP versus K1K2G3 channels fused to GFP (Clontech, CA), images of sections from oocytes expressing the different channels were recorded using the same values for parameters such as laser intensity, pinhole, and offset.
Heterologous coexpression of the GIRK1 and KGP channel subunits in Xenopus oocytes produces greatly enhanced currents (8). Comparison between the GIRK1- and KGP-deduced protein sequences revealed 66% identity or 92% similarity in a central region (region 2), whereas little similarity was found between either the N- (region 1) or C-terminal (region 3) regions of the two proteins (Fig. 1A). We will refer to each of the three regions of the two proteins using the appropriate subscript numbers (1, 2, and 3) preceded by a "G" for GIRK1 or a "K" for KGP. We hypothesized that the coexistence of the nonhomologous regions between the two proteins could be involved in the current enhancement seen upon their assembly. Fig. 1B describes the nomenclature of the specific chimeric and deletion constructs between GIRK1 and KGP used to test this hypothesis.
Because the C terminus of GIRK1 has been implicated in gating
interactions of recombinant inwardly rectifying K+ channels
(26), we tested the involvement of the G3 region (region 3 of GIRK1) in affecting the basal currents of heteromeric K+
channels. The effects of sequential deletions of the G3
region of GIRK1 (GIRK11, GIRK1
2, and
G1G2) on current size resulting from
coexpression with KGP were assessed. Xenopus oocytes were injected with GIRK1, KGP, GIRK1/KGP, or the G3 deletion
constructs of GIRK1 expressed alone or in combination with KGP cRNAs,
and Ba2+-sensitive membrane currents were measured using
two-electrode voltage clamp. Fig. 2A shows
representative Ba2+-sensitive current traces and
corresponding mean current-voltage relationships from such an
experiment. Fig. 2B summarizes in bar graph form data
comparing current magnitudes from oocytes injected with GIRK1 or its
C-terminal deletion constructs alone or together with KGP. Incremental
deletions of the G3 region of GIRK1 in the GIRK1/KGP
heteromer resulted in corresponding decreases in the magnitude of the
enhanced inwardly rectifying current. These results suggested the
involvement of the G3 region in the enhancement of the
heteromeric GIRK1/KGP channel currents.
A Chimera between GIRK1 and KGP Yields Large Inwardly Rectifying Currents
We proceeded to construct chimeras that exchanged the
nonhomologous C-terminal ends of the two channel subunits
(i.e. K1K2G3 and
G1G2K3) in order to test the
ability of these regions (G3 or K3) to produce
large currents as parts of a homomeric complex of a single protein.
Oocytes were injected with GIRK1, KGP, GIRK1/KGP, K1K2G3, or
G1G2K3 cRNAs, and
Ba2+-sensitive currents were measured. The
K1K2G3 chimera yielded large
inwardly rectifying currents (Fig. 3A). In
contrast, expression of the G1G2K3
chimera alone gave small currents. These results indicate that regions
represented by the K1K2G3 chimera
can produce large currents, suggesting that the presence of
G3 or possibly the absence of K3 plays an
important role in the enhanced currents of heteromers. Because of the
small currents displayed by the G1G2K3 chimera, we further tested
its functional expression by coinjections with either the wild-type
GIRK1 or KGP subunits. As shown in Fig. 3A, the
G1G2K3/KGP coinjection produced
large currents, ensuring that the
G1G2K3 chimera produced a
functional protein. Interestingly, because the enhanced currents
obtained with the G1G2K3/KGP
coinjection do not involve the G3 region of GIRK1, regions
of GIRK1 other than G3 are also involved in producing the
large currents of the heteromeric channel.
Fig. 3B shows immunoprecipitations from oocyte membranes of metabolically labeled recombinant channel subunits using antibodies directed against either KGP or GIRK1 (8). The 45-kDa band corresponded to KGP (lane 1), whereas the 56-58-kDa doublet corresponded to GIRK1 (lane 2). Both channel subunits could be coprecipitated by either antibody (as for example by the antibody directed against KGP (lane 3)) when coexpressed in oocytes, as shown previously (8). Immunoprecipitation of the chimera K1K2G3 with either antibody gave a single ~60-kDa band, as expected from expression of a homomeric protein (lanes 4 and 5).
Ba2+-sensitive current traces and corresponding current-voltage relationships of the K1K2G3 chimera and the GIRK1/KGP heteromer are shown in Fig. 3C. Currents of the K1K2G3 chimera showed faster activation kinetics than the heteromeric wild-type currents. Thus these experiments demonstrated that homomers of the K1K2G3 chimera were capable of producing enhanced basal currents with activation kinetics similar to those of KGP.
Coexpression of the K1K2G3 chimera
with m2 receptor enabled ACh-induced activity that was pertussis
toxin-sensitive (Fig. 3D). Similarly, coexpression with
G subunits resulted in stimulation of
K1K2G3 chimeric currents. These
results indicated that the regulation of the
K1K2G3 chimera by G proteins
resembled that of the wild-type subunits (8).
To test
whether the large currents observed with the
K1K2G3 chimera were due to a
release of inhibition exerted by the K3 region, we replaced
the K3 region with I3, the corresponding region from the related but not G protein-gated channel, IRK1 (Fig.
4A). Control experiments showed that
coinjection of IRK1 with either GIRK1 or KGP did not further increase
the already high IRK1 current levels (data not shown). Oocytes injected
with the K1K2I3 chimera gave rise
to small currents comparable in size with those obtained with the
wild-type channel subunits, GIRK1 and KGP (Fig. 4B). Further
functional tests for expression of the
K1K2I3 chimera involved its
coinjection with the wild-type GIRK1 subunit. Coinjection of the
K1K2I3 chimera with GIRK1 produced
greatly enhanced currents, ensuring that the
K1K2I3 chimera produced a
functional protein (Fig. 4B). Thus, the results from these
experiments suggest that it is not the absence of the K3
region that is responsible for the large currents seen with the
K1K2G3 chimera.
The G3 Region of GIRK1 Is Involved in Producing the Large K1K2G3 Chimeric Currents
Sequential deletions of the G3 region in the
K1K2G3 chimera were performed
(i.e. K1K2G31,
K1K2G3
2, and
K1K2) to assess the effects of G3
on the magnitude of the currents. Note that the
1 and
2 deletions
of G3 in the K1K2G3
chimera were identical to those presented earlier for GIRK1 (Fig.
2B). Fig. 4C shows current magnitudes from
oocytes injected with the K1K2G3
chimera or its C-terminal deletion constructs. Incremental deletions of the G3 region in the context of the
K1K2G3 chimera resulted in corresponding decreases in the magnitude of the current. Further functional tests for expression of the K1K2
deletion construct, which showed small currents, ensured that
K1K2 produced a functional protein, because
coexpression with GIRK1 resulted in enhanced currents. These results,
taken together with those presented in Fig. 2, demonstrate the crucial
role of the G3 region of GIRK1 in generation of enhanced
currents both in the K1K2G3
chimera, as well as in the GIRK1/KGP heteromer. Moreover, the similar
pattern of the effect of the sequential deletions on both enhanced
currents in the chimera or the heteromer suggests that elements
throughout the length of the G3 region are important
determinants of the large current size.
Because the absence
of the K3 region of KGP did not prevent constructs
containing the K1 and K2 regions from forming
active heteromeric channel currents with GIRK1 (Fig. 4, B
and C), we proceeded to test the involvement of the
K1 and K2 regions in current enhancement. We
constructed two additional chimeras,
K1G2G3 and
G1K2G3, to separate each of the two
KGP regions from one another and to test them in the context of GIRK1
regions. Oocytes injected with the
K1G2G3 chimera did not give rise to
large currents (Fig. 5A). Coinjection of the
K1G2G3 chimera with the KGP (but
not with the GIRK1) subunit produced greatly enhanced currents,
ensuring that this chimera gave rise to a functional protein (Fig.
5A). These experiments suggest that the K1
region of KGP is not sufficient to produce enhanced currents both in
chimeras and in the heteromer with GIRK1. In contrast, oocytes injected
with the G1K2G3 chimera did give
rise to sizable currents (Fig. 5B). In addition
coinjection of the G1K2G3
chimera with GIRK1 resulted in greatly enhanced currents, compared with
the currents of the G1K2G3 chimera
alone (Fig. 5B). Because K2 was the only KGP
region present in this coexpression experiment, we concluded that
K2 is sufficient to enhance currents in the heteromeric
channels. Interestingly, the enhanced currents of the
G1K2G3 chimera were much smaller
than those of the K1K2G3 chimera,
consistent with the notion that the presence of the N terminus of GIRK1
may be somehow decreasing basal currents (27). Single-channel recording
showed that G1K2G3 had conductance
and mean open time similar to KGP and an NPo in between that of KGP and K1K2G3
(data not shown).
Single-channel Characteristics of K1K2G3 Reveal a Higher NPo than That of KGP
Single-channel recordings of the
K1K2G3 were obtained and compared
with the corresponding wild-type KGP and GIRK1/KGP channel activities.
Cell-attached recordings from oocytes expressing wild-type KGP or
chimeric K1K2G3 channels displayed
similar open time kinetics, distinct from those obtained by the
GIRK1/KGP coinjections (Fig. 6). Although single-channel
activity was highly variable, the activity (NPo)
trend obtained from cell-attached patches expressing KGP,
K1K2G3, or GIRK1/KGP was similar to
the corresponding whole-cell current amplitudes. The single-channel
conductance of K1K2G3 was similar
to that previously shown for KGP (8). These data suggest that the
structural determinants represented in the
K1K2G3 chimera show enhanced
unitary activity with KGP-like characteristics.
K1K2G3 and KGP Proteins Show Similar Surface Expression in Oocyte Membranes
To investigate
whether there was an increase in the amount of
K1K2G3 chimeric proteins expressed
on the oocyte cell membrane compared with that of KGP, we fused the GFP
to the C-terminal ends of both channels and examined the surface
protein level by fluorescence confocal microscopy. Uninjected oocytes
gave very weak fluorescence background (Fig.
7A). Oocytes injected with 2 ng of KGP-GFP
RNA showed strong fluorescence localized in the plasma membrane (Fig.
7B). Injection of a 5-fold higher concentration of KGP-GFP
RNA resulted in an apparent increase in the amount of fluorescence
detected on the cell membrane (Fig. 7C). Injection of
corresponding amounts of K1K2G3-GFP
RNA resulted in qualitatively similar fluorescence signals on the cell
surface as with KGP-GFP (Fig. 7, D and E). Both
fusion proteins produced similar macroscopic currents (Fig.
7F) and single-channel characteristics (data not shown)
compared with the parental counterparts, indicating that the 238-amino
acid-long GFP does not interfere with normal channel function.
Moreover, the increase in fluorescence on the oocyte surface correlated
well with the larger magnitude of the basal currents recorded from
oocytes injected with increasing amount of
K1K2G3-GFP RNA (Fig.
7F). Hence, there was no qualitative difference on the
surface expression of the two channel proteins when injected in
comparable amounts in Xenopus oocytes. Yet 2 ng of
K1K2G3-GFP produced much greater
currents than 10 ng of KGP-GFP despite the inverse relationship on
surface expression, suggesting that the K1K2G3 channels were more active
compared with the wild-type KGP channels. These results, together with
those obtained from the single-channel measurements, suggest that it is
the frequency of opening of K1K2G3
that must be higher than that of KGP, because the two channels have
similar mean open times.
We investigated the role of the homologous and nonhomologous regions between GIRK1 and KGP in the current enhancement seen upon coexpression of the two subunits, using deletion mutants and chimeric constructs between the two proteins. We present evidence that the G3 region of GIRK1 is involved in the current enhancement of KGP currents. Although the G3 region is involved in enhancing KGP currents, its presence was not absolutely necessary, because enhanced currents could result even in its absence (as were seen when G1G2K3 and KGP were coexpressed). This result is consistent with a previous report concluding that the GIRK1 segment from amino acids 357 to 501 (same as the G3 region) was not needed for CIR-dependent agonist-induced current enhancement (29). This result is also consistent with our previous finding that a single P-region residue, Phe137 of GIRK1 (in the G2 region), is also involved in enhancing KGP current and is responsible for the prolonged single-channel openings of the heteromer (17). K2, on the other hand, was the only single region of KGP that together with GIRK1 regions produced large currents, as could be demonstrated with the G1K2G3 chimera alone or its coexpression with GIRK1. The necessity of the presence of K2 was suggested by all cases where current enhancement was observed.
The mechanism by which the G3 and K2 regions
produce enhanced currents is not clear. The gating mechanism of the
heteromeric channel is undoubtedly complex and presently not fully
understood. On one hand, the channel can be gated by a mechanism
independent of G protein activation that involves a functional
modification dependent on MgATP followed by intracellular
Na+ gating (30). It is possible that direct interactions of
the G3 and K2 regions in the context of a
tetrameric channel result in large basal currents, independent of G
protein gating. On the other hand, the G subunits of
G proteins have been shown to directly bind to both GIRK1 and CIR
channels (31), to the C termini of both GIRK1 (32) and CIR (or GIRK4)
(33), or to specific regions within the C terminus of GIRK1 (29, 34). Moreover, it has also been demonstrated (6, 35) that
G
subunits stimulate recombinant heteromeric GIRK
subunit activity in a manner similar to that of the native ACh-induced
K+ channel (1). In addition, pertussis toxin treatment of
GIRK1/KGP heteromeric or KGP homomeric channels reduced basal and
abolished agonist-induced currents, suggesting that channel gating
proceeds to a large extent, but not exclusively, via a pertussis
toxin-sensitive G protein (8). Thus, although the
K1K2G3 chimera remains sensitive to
G protein regulation, it is possible that it brings together regions of
the two subunits that alter the number or quality of G
-binding sites, thus fine tuning its response to
endogenous or to overexpression of exogenous G
subunits. Such a complicated scenario will require testing by further
detailed studies. It has been previously shown that the region of the C terminus encoded between Lys405 and Thr501 does
not bind G
subunits (34). Our GIRK1
2 construct, which lacked the segment from Arg409 to Thr501,
exhibited greatly reduced currents when coexpressed with KGP. This
result would argue against the possibility that the current enhancement
seen in the heteromeric or chimeric channels is controlled by the mere
presence of G
-binding sites. A recent study has
implicated the proximal C-terminal region of IRK1 region between amino
acids 220 and 300) as partly responsible for the assembly of inwardly
rectifying potassium channels (36). GIRK1, GIRK4, and IRK1 show high
homology in a region spanning amino acids 50-350. If GIRK channels use
this same region as other inwardly rectifying K+ channels
for assembly, then the effects of the G3 region on KGP activity are unlikely to be due to an enhanced assembly of the individual subunits.
The full-length C-terminal cytoplasmic tail (amino acids 183-501) (26) has been shown to block GIRK1 and ROMK1 macroscopic currents in Xenopus oocytes. Our data on deletion mutants of both GIRK1 and K1K2G3 chimera in the G3 region (amino acids 359-501) did not result in an increase in macroscopic current as would be expected from a release of physical block on the channel. These results would suggest that the sequence constituting the physical block proposed by Dascal et al. (26) does not lie in the last 150 amino acids of GIRK1.
The activation kinetics of the K1K2G3 or G1K2G3 chimeras were faster than those of the currents of the GIRK1/KGP heteromers, resembling those of KGP alone (8). Similarly, single-channel characteristics of the active chimeras K1K2G3 or G1K2G3 resembled those of KGP in open-time kinetics and conductance. These results suggest that the K2 region contains the structural determinants responsible for these KGP channel characteristics. The increase in the magnitude of the whole-cell current of the K1K2G3 or G1K2G3 chimeras correlated well with the increase in single-channel activity (NPo). Yet, this increase in NPo did not include a contribution from a change in the mean open time for the chimeras as it did for the heteromers. These observations suggest that region 2 of the subunits could determine open times (K2 promoting short openings, whereas G2 promoting longer openings), whereas region 3 (or its regulators) could modulate the frequency of channel opening. This scheme is consistent with the notion that Phe137 of GIRK1 controls the gating kinetics of other channel family members with which it associates (17, 37).
We thank Amanda Pabon for carrying out the immunoprecipitation experiment, Xiaying Wu for technical assistance, Dr. William Thornhill for the GIRK1 antibody and IRK1 clone, and Dr. Mika Yoshida for advice on the use of GFP and confocal microscopy. We are grateful to J. Ashot Kozak, Dr. M. Noëlle Langan, and Dr. William Thornhill for critical comments on the manuscript.