Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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A single amino acid mutation (G156S) in the putative pore-forming region of the G protein-sensitive, inwardly rectifying K+ channel subunit, GIRK2, renders the conductance constitutively active and nonselective for monovalent cations. The mutant channel subunit (GIRK2wv) causes the pleiotropic weaver disease in mice, which is characterized by the selective vulnerability of cerebellar granule cells and Purkinje cells, as well as dopaminergic neurons in the mesencephalon, to cell death. It has been proposed that divalent cation permeability through constitutively active GIRK2wv channels contributes to a rise in internal calcium in the GIRK2wv-expressing neurons, eventually leading to cell death. We carried out comparative studies of recombinant GIRK2wv channels expressed in Xenopus oocytes and COS-7 cells to determine the magnitude and relative permeability of the GIRK2wv conductance to Ca2+. Data from these studies demonstrate that the properties of the expressed current differ in the two systems and that when recombinant GIRK2wv is expressed in mammalian cells it is impermeable to Ca2+.
potassium channels; weaver mice; G proteins; Xenopus oocytes; voltage clamp
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INTRODUCTION |
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THE MURINE WEAVER DISEASE is caused by the mutation of a single amino acid (G156S) in the putative pore region of the inwardly rectifying K+ channel, GIRK2. Previous studies have shown that recombinant GIRK2wv expressed heterologously in Xenopus oocytes and mammalian cells, as well as in cerebellar granule cells from wv mice, formed homomultimeric channels characterized by 1) G protein insensitivity, 2) cation nonselectivity, and 3) sensitivity to QX314, MK-801, and verapamil inhibition.
Indirect evidence from a number of studies supports the hypothesis that
the mutant weaver channel might render neurons leaky to
Ca2+, eventually resulting in cell death. A recent study
demonstrated that intracellular Ca2+ was elevated in the
primary cerebellar neuronal cultures from heterozyogous (wv/+)
animals relative to wild type (4). Silverman and colleagues (14) have
reported an apparent permeability of GIRK2wv channels to
Ca2+ over wild-type GIRK channels when expressed in
Xenopus oocytes. In the Silverman et al. study, the
Ca2+ permeability of the channel was inferred from the
activation of the endogenous Ca2+-activated
Cl conductance, which was seen only in
GIRK2wv-expressing oocytes (14). In parallel studies of
recombinant GIRK2wv channels in oocytes, removal of
Ca2+ from the incubation medium was shown to significantly
enhance oocyte survival (18). Taken together, these observations
suggested that GIRK2wv channels are permeable to monovalent as
well as divalent cations.
In this study, we directly tested whether homomeric GIRK2wv channels are permeable to Ca2+ in both Xenopus oocytes and mammalian cells transiently expressing GIRK2wv channels in culture. Homomeric channels were weakly permeable to Ca2+ in the Xenopus oocytes. In contrast, the divalent cation permeability was absent in the GIRK2wv-expressing mammalian cells, suggesting that susceptibility to cell death in GIRK2wv-expressing neurons may simply be due to Ca2+ influx through parallel, voltage-dependent channels following prolonged depolarization.
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MATERIALS AND METHODS |
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cDNA clone. GIRK1 was cloned from a RIN cell library and had a predicted amino acid sequence identical to the cardiac clone originally described (8). GIRK2 and GIRK2wv were gifts from Dr. P. Kofuji (California Institute of Technology, CA). The m2 muscarinic receptor was purchased from Clontech (Clontech, CA) in the pGEM3Z vector. All GIRK constructs were subcloned into either the pMXT vector, obtained from Dr. P. Kofuji, for oocyte expression or pEGFPN3 (Clontech, CA) for mammalian expression. The m2 receptor was linearized with Hind III, and cRNA was transcribed using the T7 polymerase mMessage mMachine kit (Ambion, Austin, TX). All GIRK constructs were linearized with Sal I, and cRNA was transcribed using T3 polymerase mMessage mMachine kit (Ambion). The cRNA concentration was determined by ultraviolet light absorption at 260 nm (A260) and confirmed by intensity on ethidium bromide stained agarose gels. For mammalian cell expression, GIRK1, GIRK2, and GIRK2wv were fused to enhanced green fluorescent protein (EGFP) at the carboxy terminus in the pEGFPN3 vector (Clontech).
Oocyte electrophysiology. Oocytes were injected with 2 ng of m2 muscarinic receptor cRNA and 5 ng of each GIRK subunit cRNA along with 12.3 ng GIRK5 (KHA1) antisense cRNA. Injected oocytes were maintained in OR-2+ solution containing (in mM) 96 NaCl, 2.5 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, 2.5 sodium pyruvate, and 50 µg/ml gentamicin. Nominally Ca2+-free solutions were used to incubate and maintain oocytes expressing GIRK2wv.
Two-microelectrode voltage-clamp recordings were performed 3 days postinjection using a TURBO TEC-10C amplifier (NPI, Tamm, Germany). Data were acquired using Pulse software (HEKA, Lambrecht, Germany), an ITC-16 interface (Instrutech, Great Neck, NY), and an IBM-compatible PC. Microelectrodes were filled with 3 M KCl and had resistances of 0.5-2 MMammalian cell culture and cDNA transfection. COS-7 cells (American Type Culture Collection) were plated in 35-mm dishes and grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transfection of COS-7 cells was carried out using SuperFect Transfection Reagent (10 µl per dish; QIAGEN, Valencia, CA) mixed with the following amounts of cDNA: 0.5 µg m2R with 1 µg GIRK1-EGFP and GIRK2 or GIRK2wv-EGFP in 100 µl serum-free media (Opti-MEM1; GIBCO, Grand Island, NY). Cells were exposed to the DNA-containing solution for 10 min at room temperature, followed by the addition of 600 µl of serum-containing cell culture media. Cells were then incubated for 2 h, washed once, and incubated at 37°C, 5% CO2. Electrophysiological recordings from COS-7 cells were made 48 h from transfection initiation on green fluorescent protein positive cells.
Electrophysiological recording from mammalian cells.
Whole cell recordings were performed at room temperature
48-72 h posttransfection in an initial bath solution consisting of (in mM) 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2,
and 10 HEPES, pH 7.4. After currents in low-K+ solutions
were recorded, cells were superfused with either a high-K+
solution containing (in mM) 140 KCl, 1 MgCl2, and 10 HEPES,
pH 7.4, or a high-Na+ solution containing (in mM) 145 NaCl,
1 MgCl2, and 10 HEPES, pH 7.4. In experiments designed to
quantitate the Ca2+ permeability, cells were superfused
with 5 or 70 mM Ca2+ bath solution containing (in mM) 5 CaCl2 with 140 NMDG-Cl, or 70 CaCl2 with 35 NMDG-Cl, 1 MgCl2, 10 HEPES, pH 7.4. Solution osmolarity was
kept constant at 270-290 mosM using a vapor pressure osmometer
(Wescor, Logan, UT). Patch pipettes were pulled from microhematocrit
capillary tubes (Fisherbrand, Fisher, Pittsburgh, PA) to give
resistances of 3-6 M. The pipette recording solution contained
(in mM) 120 KCl, 2 CaCl2, 1 MgCl2, 11 EGTA, 33 KOH, 10 HEPES, 1 NaGTP, 2 MgATP, pH 7.2, or 140 NMDG, 0.2 CaCl2, 1 MgCl2, 1 EGTA, 10 HEPES, 1 NaGTP, 2 MgATP, pH 7.2 when Ca2+ currents were measured. Whole
cell currents were recorded with an EPC-7 (List Electronics, Lambrecht,
Germany) patch-clamp amplifier at 2 kHz and low-pass filtered at
1 kHz. Stimulation and data acquisition were controlled by the PULSE
software package on a Power Macintosh computer, and data analysis was
performed with IGOR software (WaveMetrics, Lake Oswego, OR).
Digital fluorescent imaging. COS-7 cells were grown on 25-mm coverslips to 50% confluence, loaded with 2 µM fura 2-AM (Molecular Probes) in serum-free MEM for 1 h, then equilibrated in Hanks' balanced saline solution (HBSS; GIBCO, Grand Island, NY) for 50 min. Fura 2 loading and equilibration were carried out at 37°C. The coverslip with loaded cells was moved into a Teflon coverslip recording dish (model LU-CSD; Medical Systems, NY) and mounted on the microscope stage. Cells were continuously superfused throughout the experiment with HBSS or switched to a 70 mM Ca2+ solution containing (in mM) 70 CaCl2 with 35 NMDG-Cl, 1 MgCl2, and 10 HEPES, pH 7.4. Internal Ca2+ (Cai) was determined using digital fluorescent imaging of cellular fura 2 epiflorescence. Emission was determined at 510 nm following excitation at 340 and 380 nm. Images were obtained every 20 s, and 64 frames were averaged at each excitation wavelength. Background was obtained using an area of the coverslip devoid of cells and subtracted from each excitation wavelength image. After background subtraction, the 340-nm image was divided by the 380-nm image to provide a ratio (R) image. Image analysis was carried out using the ImageMaster Ratio Fluorescence Imaging Software (Photon Technology International, NJ).
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RESULTS |
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Comparison of GIRK1/2 and GIRK2wv channels expressed in Xenopus oocytes and COS-7 cells. The goal of this study was to quantitate the magnitude of the Ca2+ permeability of recombinant GIRK2wv channels compared with heteromultimeric GIRK1 + GIRK2 (GIRK1/2) channels. We compared expression of the wild-type and mutant channels in Xenopus oocytes to that in mammalian COS-7 cells.
Recombinant GIRK subunits coassemble with endogenous Xenopus GIRK5 subunits to form functional channels (5). Antisense cRNA against GIRK5 (KHA1) has been previously reported to knock out endogenous GIRK5 expression in oocytes (5, 15). Therefore, antisense cRNA against GIRK5 was coinjected in all our studies to prevent endogenous GIRK5 expression and coassembly. Figure 1, A and B, compares expression of the heteromultimeric GIRK1/2 conductance in a representative Xenopus oocyte and mammalian cell. Both expression systems gave rise to carbachol-induced currents that were inwardly rectifying and Ba2+ sensitive. When expressed in oocytes, GIRK1/2 was associated with a large basal (carbachol-independent) current in high-K+ solutions. The corresponding basal current was absent in the COS-7 cells. Average peak current amplitude of the carbachol-sensitive K+ current was
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Divalent permeability of uninjected oocytes.
To quantitate the magnitude of the GIRK2wv-induced
Ca2+ influx pathway, it was necessary to identify basal
divalent permeability through endogenous, voltage-gated
Ca2+ channels in uninjected oocytes exposed to solutions
containing elevated divalent concentrations. Characterization of
endogenous voltage-activated Ca2+ channels in oocytes has
been previously established (2, 9, 12). A comparative summary of the
magnitude of both inward and outward current at 150 and 50 mV,
in high and low Ca2+ solutions is plotted in Fig.
3 as a function of internal
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffering. Charnet and colleagues (1) have previously reported the use of BAPTA injections to buffer the influx of
Ca2+ in oocytes, thereby preventing activation of the
endogenous Ca2+-activated anion conductance.
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Direct measurement of current carried by
Ca2+ in oocytes and mammalian
cells expressing GIRK1/2 and GIRK2wv.
To determine the magnitude of divalent current carried by
GIRK2wv channels compared with GIRK1/2 channels, experiments on both oocytes and COS-7 cells were performed in solutions in which Ca2+ was the only permeant cation in the extracellular
solution. Oocytes were injected with 100 mM BAPTA prior to current
recording in high divalent solutions to block activation of the
contaminating Ca2+-activated Cl current.
Figure 4 illustrates data obtained from
both oocytes and COS-7 cells expressing GIRK1/2. Both basal and
carbachol-induced K+ currents were recorded to ensure that
cells were expressing G protein-activated GIRK channels. Sequential
exposure of COS-7 cells to 5 and 70 mM Ca2+-containing
solutions failed to result in current activation. Current amplitudes at
150 mV for oocytes showed no significant change on switching
from low to high external Ca2+.
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Intracellular calcium measurements.
To further investigate whether constitutively active
GIRK2wv channels expressed in mammalian cells could give rise
to significant changes in levels of intracellular calcium
(Cai), as has been reported for neurons cultured from
weaver mice (4, 21), we carried out digital fluorescent imaging
experiments in COS-7 cells transfected with GIRK1/2 or GIRK2wv
where GIRK1 and GIRK2wv were tagged with EGFP. Fura 2 was used
to detect resting Cai in nontransfected COS-7 cells, COS-7
cells cotransfected with GIRK1-EGFP, and GIRK2 or COS-7 cells
transfected with GIRK2wv-EGFP. Cells were loaded with fura 2-AM
for 1 h before digital fluorescent imaging experiments. The resting
340/380 ratios (R) among the three groups were indistinguishable. These
data are summarized in Fig. 7. The dynamic
range of the cellular response to changes in Cai was
determined in experiments in which cells were sequentially exposed to a
solution containing 10 µM ionomycin in the presence of 2 mM
Ca2+, followed by a solution change to one in which the
free Ca2+ concentration was buffered to zero in the
presence of 1 mM EGTA as seen in Fig. 7A. The mean resting
340/380 nm fluorescence intensity ratio (R340/380) values
were 0.6 ± 0.02 (n = 43) for nontransfected cells, 0.55 ± 0.09 (n = 32) for GIRK1/GIRK2-expressing cells, and 0.57 ± 0.03 (n = 34) for the GIRK2wv-expressing cells (Fig. 7B). We were unable to detect a change in the
R340/380 values on changing from low (2 mM) to high (70 mM)
external Ca2+ in either the GIRK1/2- or
GIRK2wv-transfected cells. The average of the change in the
R340/380 in individual cells on increasing extracellular
Ca2+ from 2 to 70 mM was 0.28 ± 0.02 (n = 5 ) for
GIRK1/2 and 0.21 ± 0.03 (n = 9) for GIRK2wv as
summarized in Fig. 7C.
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DISCUSSION |
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Expression and activation of GIRK2wv in oocytes has been
associated with a large increase in the endogenous
Ca2+-activated Cl current, indicative of
a large divalent influx in Ca2+-containing solutions (14).
This observation, along with the increased vulnerability of oocytes to
cell death in Ca2+-containing solutions (18), has suggested
that the GIRK2wv channel might allow for a significant
Ca2+ "leak." These observations prompted our
investigation of the magnitude of the GIRK2wv divalent
permeability in heterologous expression systems.
In this study, we have compared expression of GIRK1/2 and
GIRK2wv channels in both oocytes and mammalian cells. We
compared the divalent permeability of the wild-type to the mutant
GIRK2wv channels and found that oocytes expressing the mutant
channels show 1) an elevation of inward current over those
expressing the wild-type GIRK1/2 and 2) a shift in reversal
potential on switching from low- to high-Ca2+-containing
external solutions. These results were obtained in the presence of high
concentrations of internal BAPTA to inhibit activation of contaminating
Ca2+-activated Cl currents. Similar
experiments in transfected COS-7 cells failed to demonstrate either a
significant increase in Ca2+ current through the mutant
channels over that observed in GIRK1/2-expressing cells or a shift in
reversal potential on increases in extracellular Ca2+. We
were also unable to observe any significant increase in the resting
levels of Cai in COS-7 cells expressing GIRK2wv
over that observed in nontransfected cells or cells expressing GIRK1/2. Taken together, these data indicate that the divalent cation properties of the expressed GIRK2wv conductance differ between the two
heterologous expression systems. Most importantly, however, the data
strongly suggest that, if the mammalian cells are a better model for
channel expression in mouse neurons than the oocytes, the elevated
Cai levels observed in weaver neurons (4, 21) may
be a result of chronic depolarization and not Ca2+ influx
through the GIRK2wv channels themselves. Evidence in support of
this hypothesis comes from the studies of Liesi and Wright (10) who
demonstrated that Ca2+ channel function is essential in
mediating the weaver gene effect. The rescue effect of
weaver granule cell neurons at high but not low concentrations
of MK-801 in the studies of Liesi and Wright (10) was consistent with
the reported inhibitory effect of verapamil and high concentrations of
MK-801 on voltage-gated Ca2+ channels; low concentrations
of MK-801 (1 µM) had no rescue effect (10).
Determination of the Ca2+ permeability of the homomultimeric GIRK2wv channel is more direct in mammalian cells lacking the contaminating endogenous Ca2+-activated anion conductance. In our experiments, we found no evidence for Ca2+ flux through GIRK2wv channels monitored as either an increase in inward current in high divalent solution or in measurements of changes in resting Cai as monitored by ratiometric intracellular fura 2 fluorescence. Consistent with the previous anecdotal observation made by Navarro and co-workers (13), we were unable to observe an increase in inward current in Chinese hamster ovary (data not shown) as well as COS cells transfected with the GIRK2wv gene in 70 mM Ca2+ containing external solutions.
It is tempting to generalize that the weaver mutation in the signature sequence of all K+ selective channels would produce a similar loss in K+ selectivity in the outwardly as well as the inwardly rectifying K+ conductances. Interestingly, this same mutation has been found in a member of the six-transmembrane family of K+ channels, which points to the contrary. The K+ channel, KCNQ4, localizes its expression to cochlear outer hair cells and maps to the DFNA2 locus for a form of nonsyndromic dominant deafness (7). A mutation in this gene in the DNFA2 pedigree exchanges the G for an S (G285S) in the GYG sequence in the pore of that channel, identical to the mutation in GIRK2wv. The G285S mutation in KCNQ4 exerts a strong dominant negative effect on wild-type KCNQ4, and its loss leads to slow cellular degeneration (7), although the precise pathogenesis is unknown. KCNQ4 codes for a six-transmembrane domain K+ channel subunit protein that is assumed to form a functional heterotetramer with other members of the KCNQ family. Unlike GIRK2wv, which has an equivalent mutation in the signature sequence, the mutation G285S in KCNQ4 does not appear to form functional homomultimers as does GIRK2wv. Coexpression studies with the mutant KCNQ4 G285S and other members of the KCNQ family carried out to date show that coexpression of the mutant subunit reduces current expression by ~90%. The remaining current is K+ selective over Na+ or Ca2+ (7), unlike the selectivity profile of the mutant GIRK2wv. Thus similar pore mutations in the outward and inwardly rectifying K+ channel families would appear to have significantly different functional phenotypes with respect to changes in channel selectivity and ability to form functional homo- and heteromultimers (6, 16). The two transmembrane domain GIRK subunits appear to tolerate changes in pore-forming residues allowing for the formation of hetero- as well as homomultimeric channels. The six-transmembrane domain K+ channels appear to require a more rigid scaffolding intolerant of similar changes in pore-forming residues.
In addition to the observed differences in selectivity between GIRK1/GIRK2 heteromultimers and GIRK2wv homomultimers, we observed a consistent difference in the kinetics of current activation for the two channels at the most hyperpolarized potentials when expressed in Xenopus oocytes. Current activation for GIRK2wv expressed in oocytes was instantaneous, whereas the kinetics of activation for GIRK1/GIRK2 were much slower. Similar differences in the time course of current activation on hyperpolarization between recombinant GIRK1/GIRK2 and GIRK2wv channels have been observed by Slesinger et al. (16). Differences in time course of current activation between the wild-type GIRK1/GIRK2 channels and the mutant GIRK2wv channels were not observed when the recombinant channels were expressed in the mammalian cell background. These differences in activation kinetics, which we observed exclusively in the oocyte expression experiments, are consistent with the weak inward rectification of the currents also observed for the mutant GIRK2wv channel in the oocyte system in our studies as well as those of Kofuji et al. (6). Rectification in the inwardly rectifying K+ channels is a result of Mg2+ and/or polyamine binding to an intracellular site, thereby blocking monovalent cation permeation in the outward direction (3, 11, 17, 19). It may be that the reduced rectification seen for GIRK2wv when expressed in oocytes may be due to weak binding and/or permeation of a class of cytoplasmic polyamines not present in the mammalian cells.
In summary, results for our investigation indicate that the modest Ca2+ influx through GIRK2wv homomeric channels expressed in oocytes differs from that observed for channels expressed in mammalian cells and may represent the formation of a functional channel arising from coassembly with an unidentified endogenous subunit of the oocyte. Coassembly with the endogenous Xenopus oocyte subunit GIRK5 is unlikely, in that our experiments were conducted using antisense against GIRK5, which would have prevented its expression. Recombinant GIRK2wv channel expression in mammalian cells was not associated with either an observable Ca2+ permeation through the conductance nor an increase in intracellular Cai over that observed in nontransfected cells. Our data suggest that the elevation in Cai associated with neuronal cell death in murine cells expressing the gene may not be due to divalent permeation through the GIRK2wv homomeric channels but may be due instead to toxicity induced through chronic depolarization, allowing for Ca2+ influx through voltage-dependent Ca2+-permeable pathways.
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ACKNOWLEDGEMENTS |
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We thank Drs. Aaron P. Fox, G. Breitwieser, and D. A. Hanck for many helpful discussions as well as Boris Krupa for technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of General Medical Sciences Grants RO1 GM-36823 and RO1 GM-54266.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. J. Nelson, The Univ. of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, MC0926, 947 East 58th St., Chicago, IL 60637 (E-mail: dnelson{at}drugs.bsd.uchicago.edu).
Received 30 April 1999; accepted in final form 21 December 1999.
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