From the Division of Cardiology and Cardiovascular Research Laboratory, UCLA School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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Muscarinic potassium channels are heterotetramers of Kir3.1 and other Kir3 channel subunits and play major roles in regulating membrane excitability in cardiac atrial, neuronal, and neuroendocrine tissues. We report here that rabbit atrial muscarinic potassium channels are rapidly and reversibly inhibited by membrane stretch, possibly serving as a mechanoelectrical feedback pathway. To probe the molecular basis for this phenomenon, we heterologously expressed heteromeric Kir3.1/Kir3.4 channels in Xenopus oocytes and found that they possess similar mechanosensitivity in response to hypo-osmolar stress. This could be attributed in part, if not exclusively, to the Kir3.4 subunit, which reproduced the mechanosensitivity of the heteromeric channel when expressed as a homomeric channel in oocytes. Kir3.4 is the first stretch-inactivated potassium channel to be identified molecularly. Physiologically, this feature may be important in atrial volume-sensing and other responses to stretch.
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INTRODUCTION |
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Although excitation-contraction coupling is the major mechanism
regulating cardiac function, mechanoelectrical feedback plays important
modulatory roles (1). Mechanoelectrical feedback is particularly
essential in the atria of the heart, which regulate vascular volume
through secretion of atrial natriuretic peptides when atrial myocytes
are stretched. A number of mechano-sensitive ion channels have been
identified in atrial tissue, including stretch-activated potassium,
chloride, and nonselective cation channels (2-6) and
stretch-inactivated potassium channels (7). However, the molecular
identities of these channels are currently unknown. Since cardiac
muscarinic potassium channels
(KACh)1 (8, 9)
regulated by G proteins (10) are preferentially expressed in atrial tissues (11), they seemed likely candidates to
examine for mechano-sensitive properties. Moreover, they have been
characterized at the molecular level as heterotetramers of Kir3.1
(GIRK1) and Kir3.4 (GIRK4) proteins (12).
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MATERIALS AND METHODS |
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Atrial Myocyte Isolation and Current Recording--
Rabbit
atrial myocytes were isolated enzymatically and patch-clamped in the
whole-cell recording configuration as described previously (13, 14).
Calibrated positive pressure was applied to myocytes through a
water-filled U-tube connected to the patch electrode. Application of
positive pressure (10 cm of H2O) did not significantly
change the series resistance (6.2 ± 0.9 to 5.9 ± 0.8 megaohms, n = 9). Patch pipettes (resistance 0.2-0.5
megaohms) contained 150 mM KCl + KOH, 5 mM
NaCl, 1 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, pH 7.2, and the bath
solution contained 150 mM NaCl + NaOH, 10 mM
KCl, 1 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, 0.02 mM tetradotoxin, 0.01 mM nifedipine, pH 7.4. The calculated
potassium equilibrium potential was 65 mV.
cRNA Synthesis and Current Recording from
Oocytes--
Full-length cDNA encoding the Kir3.4 protein from a
rat brain library (confirmed by sequencing) was subcloned into
pBlueScript (Stratagene, San Diego, CA), and cRNA was made using
standardized in vitro methods (Ambion, Austin, TX). The
coding region was subcloned such that the 5 end had a Kozak sequence
and the 3
end had a poly(A) tail. Xenopus laevis oocytes
were isolated by enzymatic digestion (2 mg/ml collagenase). Stage IV-V
oocytes were used for injection. Current usually became detectable
24 h after injection, and experiments were carried out between 24 and 96 h afterward. Whole-oocyte currents were recorded at room
temperature with the two-electrode voltage clamp technique (15) using a
Dagan (Minneapolis, MN) CA-1 oocyte clamp amplifier, a TL-1 DMA
interface for data acquisition and pCLAMP software (Axon Instruments,
Foster City, CA). Recording electrodes were pulled from borosilicate
pipette glass (A-M Systems Inc., Seattle, WA) and filled with 3 M KCl. Capacitance and leak currents were subtracted after
blocking K+ currents with 5 mM
BaCl2. The standard bath solution contained 98 mM KCl, 2 mM KOH, 1.8 mM
CaCl2, 1.0 mM MgCl2, and 5.0 mM HEPES, pH 7.2. For the experiments measuring reversal
potentials, KCl was replaced isotonically by NaCl. Giant cell-attached
patches were formed on de-vitellinized oocytes (16, 17), and currents were recorded under voltage clamp conditions as described previously (17). The bath and pipette solutions (room temperature) contained 98 mM KCl, 2 mM KOH, 1.8 mM
CaCl2, 1.0 mM MgCl2, and 5.0 mM HEPES, pH 7.2.
Hypo-osmotic Challenge-- Currents were first recorded using the two-electrode voltage clamp technique in oocytes superfused with bath solution containing 50 mM KCl and 100 mM sucrose. Hypo-osmotic challenge (50%) was induced by removal of sucrose for 15-30 min. Leak current was subtracted after blocking K+ current with 5 mM BaCl2.
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RESULTS |
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Isolated rabbit atrial myocytes were patch-clamped in the
whole-cell configuration, and whole-cell currents were either recorded at a steady holding potential of 100 mV (Fig.
1a) or during periodic voltage
ramps from
100 to
20 mV (0.1 mV/ms) with Na+ and
Ca2+ currents blocked (Fig. 1b). After
potentiating the inward potassium current by exposing the myocyte to 10 µM carbachol, 10 cm of H2O positive pressure
was applied to the patch pipette to stretch the cell membrane. Positive
pressure caused a rapid (within 500 msec) decrease in the
carbachol-sensitive current, averaging 15 ± 3% at
100 mV in 5 myocytes (p < 0.05) (Fig. 1d). Upon
withdrawal of positive pressure, the current recovered rapidly and
completely. Both before and after the application of positive pressure,
current was fully blocked by 5 mM external
Ba2+. Additionally, positive pressure did not induce any
comparable changes in current before carbachol or in the presence of 5 mM extracellular Ba2+ (Fig. 1c), and
current-voltage relationship was not shifted, only reduced in amplitude
(Fig. 1b). These data suggest that the affected current
under these conditions was predominantly IK,ACh and unlikely to be due to an artifact or an endogenous
mechano-sensitive current. The inactivation was rapid (within 500 ms),
and the current recovered completely and similarly fast upon the
removal of positive pressure after 5 s. Decreases in
IK,ACh were also observed with application of as
little as 2 cm of H2O positive pressure but developed more
slowly and were not fully reversible.
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Since atrial G-protein-regulated potassium channels are known to be
heteromeric proteins composed of Kir3.1 and Kir3.4 subunits (12), we
determined whether Kir3.1/3.4 currents exhibited mechanosensitivity when heterologously coexpressed in Xenopus oocytes. To
maximally activate the channels, Kir3.1 and Kir3.4 were coexpressed
with an excess of G subunits (by injecting cRNA for
G
1 and G
2 at a 23:1 excess), which
produced a 12-fold increase in current compared with expression of
Kir3.1/3.4 without G
subunits (data not shown) when
measured with the two-electrode voltage clamp technique. To test for
mechanosensitivity, we examined the effects of reducing osmotic
pressure of the bath perfusate on the amplitude of whole-oocyte
Kir3.1/3.4 currents. A 50% reduction of osmotic strength of the bath
solution to induce oocyte swelling and membrane stretch reversibly
decreased heteromeric Kir3.1/3.4 currents measured at
100 mV by
18 ± 6% (n = 4) (Fig.
2, a and b). The
full decrease took about 10 min to develop, comparable to the time
course of osmotic swelling documented in previous studies in oocytes
(18) and was reversible over a similar time course upon restoring
normal osmolarity (Fig. 2, a and b). Under both
normal and hypo-osmolar conditions, the recorded currents were fully
blocked by 5 mM extracellular Ba2+ (data not
shown), ruling out artifact from contamination by endogenous mechanosensitive channels previously described in oocytes (19, 20). In
addition, noninjected oocytes showed no currents of comparable
magnitude under normal or hypo-osmolar conditions (data not shown). As
additional controls, we also tested the effects of hypo-osmolar
challenge in oocytes expressing either Kir1.1 (ROMK1) or Kir2.1 (IRK1)
channels. In neither case did the magnitude of current change in
response to hypo-osmolar challenge (Fig. 2, c-e),
suggesting that the cell swelling-induced inhibition of Kir3.1/Kir3.4
channels is specific to Kir3 versus other families of Kir
channels.
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To determine which Kir3 subunit was responsible for conferring
mechanosensitivity to the heteromeric channel, we attempted to express
homomeric Kir3.1 or Kir3.4 channels. As reported previously (21),
expression of Kir3.1 channels with or without G subunits produced only small currents, probably representing
heteromeric channels formed by Kir3.1 combining with endogenous Kir3.5
(XIR) subunits present in the oocytes (22). Most previous studies have
also reported that Kir3.4 channels form homomeric channels only poorly
(11, 12, 23, 24). However, by modifying the 5
- and 3
-untranslated
regions (see "Materials and Methods"), we consistently measured
large currents, typically ranging from
20 to
40 µA at
100 mV
with 100 mM potassium in the bath perfusate from
Xenopus oocytes expressing Kir3.4 alone, as assayed by the two-electrode voltage clamp (Fig. 3,
a and b). The magnitude of the current increased
linearly with the amount of cRNA injected in batches of oocytes from
the same frog on the same day (Fig. 3c). The failure of the
current magnitude to saturate as well as its large magnitude compared
with small endogenous currents in uninjected oocytes (
0.13 ± 0.23 µA at
100 mV, n = 8) argues strongly against
the possibility that Kir3.4 proteins were combining with an endogenous
Xenopus protein such as Kir3.5 (XIR) (22) to form
heteromeric channels. With 100 mM potassium in the bath solution, the large inward currents showed no or mild relaxation, and
outward currents were minimal at potentials positive to the potassium
equilibrium potential, typical for strong inward rectifier potassium
channels (25) (Fig. 3, a and b). The current was
highly selective for potassium over sodium and blocked in a
voltage-dependent manner by extracellular cesium and
barium, with Kd values at
60 mV of 61 ± 3 µM (n = 4) and 92 ± 13 µM (n = 4) respectively (data not shown).
Unitary current amplitudes at different voltages obtained from
all-points histogram analysis revealed a single channel conductance of
33.2 ± 0.3 picosiemens (n = 4) with 100 mM K+ in the pipette solution (Fig.
3d), similar to an earlier estimate (12). In contrast to the
previous studies in which single channel openings were flickery and
more short-lived (11, 12), openings lasting 5-50 ms were commonly
observed. Like native KACh channels (26), currents through
homomeric Kir3.4 channels increased by an average of 76 ± 17% at
100 mV (n = 7) in response to 10 µM carbachol when coexpressed with the m2 muscarinic receptor
(Fig. 3, e and f), as has been noted previously
(12, 24). The effect was maximal within 1-2 min and then gradually
lessened, probably due to desensitization. Coexpression of Kir3.4 with
G
subunits (at 23:1 excess of injected
G
cRNA) also boosted Kir3.4 currents by an average of
>100-fold compared with oocytes from the same batch injected with the
same amount of Kir3.4 cRNA alone (n = 4) (Fig.
3f). These results confirm that homomeric Kir3.4 channels
are also classic G-protein-regulated potassium channels.
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In attempting to measure unitary currents through homomeric Kir3.4
channels, we noted that channels ran down very rapidly after formation
of a cell-attached patch (>50 patches). This rapid rundown precluded
single channel analysis from standard patches (electrode tip, 1-3
µm) but could be quantified in giant patches (electrode tip, 20-30
µm), with the mean time constant of rundown averaging 2.3 ± 0.6 min (n = 5 giant patches). Single channel events could
often be resolved when only a few active channels were left in the
giant patch (Fig. 3d). Patch excision always led to an
immediate disappearance of channel activity. Since the formation of a
gigaseal (or patch excision) subjects the underlying membrane to
considerable mechanical forces, these observations suggested that
homomeric Kir3.4 channels might be sensitive to membrane stretch. To
test directly for mechanosensitivity, we examined the effects of cell
swelling induced by hypo-osmotic challenge on whole-oocyte homomeric
Kir3.4 currents measured with the two-electrode voltage clamp. A 50%
reduction of osmotic strength of the bath solution reversibly caused a
27 ± 4% reduction in Kir3.4 current at 100 mV
(n = 6) (Fig. 4). This
finding indicates that the Kir3.4 subunit is responsible in part, if
not exclusively, for conferring mechanosensitivity to heteromeric
Kir3.1/3.4 channels and to the cardiac G-protein-regulated potassium
channel. Whether the Kir3.1 subunit shares similar mechanosensitive
properties is uncertain at this point.
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To examine the mechanism underlying the mechanosensitivity of Kir3.4
channels, we investigated whether membrane stretch inhibited Kir3.4
currents indirectly by an effect on G-protein signaling. First, we
tested the effects of hypo-osmolar challenge on Kir3.4 currents that
had been maximally stimulated with carbachol in oocytes coexpressing
Kir3.4 and the m2 receptor. The carbachol-stimulated Kir3.4
currents demonstrated a similar decrease in response to hypo-osmolar
challenge as under basal conditions (averaging 31 ± 4%,
n = 4) (Fig. 4d). We further examined the
effects of hypo-osmotic challenge in oocytes in which Kir3.4 homomeric
channels were coexpressed with an excess of G
subunits (23:1 excess of injected G
cRNA).
Hypo-osmotic challenge decreased current at
100 mV by 18 ± 3%
(n = 5) (Fig. 4d). These findings show that the mechanosensitivity of Kir3.4 currents remains intact over a wide
range of ambient G
levels, including a presumably saturating range. This observation makes it unlikely that fluctuations in the level of G
subunits induced by membrane
stretch cause the inhibition of current.
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DISCUSSION |
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Our results demonstrate for the first time that KACh channels in the atrium are mechano-sensitive, consistent with their participation in the volume-sensing role of this organ. Physiologically, stretch-induced inactivation of KACh channels during atrial distension would facilitate membrane depolarization and enhance excitability and could potentially contribute to a variety of stretch-induced responses, including contraction-excitation coupling, atrial natriuretic peptide release, stretch-induced arrhythmias, and/or hypertrophic gene programming.
By demonstrating that both heteromeric Kir3.1/3.4 channels and homomeric Kir3.4 channels exhibit similar mechanosensitivity as native rabbit atrial KACh channels, we provide the first molecular identity of a mammalian stretch-inactivated potassium channel, which will permit structure-function studies to characterize the molecular mechanisms involved. Interestingly, the predicted overall topological structure of Kir3 channels is similar to nonmammalian mechano-sensitive ion channels cloned from Escherichia coli and Caenorhabditis elegans (27, 28), suggesting a common structural motif for these mechano-sensitive ion channels.
The mechanism responsible for mechanosensitivity in these channels is
unclear at this point. Our findings argue against a mass action effect
of stretch on G subunits as the underlying mechanism,
since the mechano-sensitive response remained intact and of comparable
magnitude over a wide range of ambient G
levels (Fig.
4d). For the case in which oocytes were co-injected with
Kir3.4 and G
cRNA at a 1:23 ratio, we presume that this includes a saturating range of G
subunits
relative to Kir3.4 molecules, although the ratio of protein molecules
cannot be assumed to be the same as the ratio of cRNA injected. Even if
a mass action effect is unlikely, G-protein signaling might be involved
if membrane stretch inhibited the ability of G
subunits to activate the channels by an allosteric, rather than mass
action, effect. By constructing chimeric proteins between Kir3.4 and
non-G-protein-regulated Kir proteins, it may be possible to resolve
this question. Alternatively, the mechanism of mechanosensitivity may
not directly involve G-protein signaling. A recent report has
demonstrated inhibition of Kir3 currents by protein kinase C (29). It
is possible that membrane stretch-induced activation of phospholipase C
(30, 31) could in turn activate protein kinase C to inhibit the
channels. Finally, a direct interaction between Kir3.4 and cytoskeletal
elements or direct sensitivity of the channel to membrane curvature are
possible mechanisms for mechanosensitivity (32). Actin has been
implicated as a cytoskeletal transducer of mechanical force for other
mechano-sensitive channels (33) and has been shown to regulate the
function of a number of ion channels such as epithelial sodium channels
(34). Also, Kir2 channels such as Kir2.1 have been shown to link to the
actin cytoskeleton as a means of spatially localizing them at specific regions in the cell (35), although no similar consensus linkage sites
have been identified in Kir3 channels. These actin-binding protein
sites do not confer mechanosensitivity to Kir2.1 when expressed in
oocytes, however, as shown in Fig. 2d. In preliminary experiments, we were also unable to restore Kir3.4 channel
activity in excised giant inside-out patches by adding F-actin to the
cytoplasmic surface of the patch. Further studies, perhaps involving
chimeric constructs between Kir3.4 and other Kir family members, will
be required to unravel the molecular basis for stretch-induced
inactivation of these channels.
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ACKNOWLEDGEMENTS |
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We thank Dr. Henry Lester for providing the
Kir3.1 and m2 receptor clones and Dr. Lutz Birnbaumer for
the G1 and G
2 clones.
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FOOTNOTES |
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* Supported by National Institutes of Health Grants RO1 HL36729 and Specialized Center of Research in Sudden Cardiac Death P50 HL52319, by American Heart Association, Greater Los Angeles Affiliate, Grants-in-aid 1110-GI1 and 1126-GI1 and Initial Investigator Award 1136-FI1, and by the Laubisch and Kawata Endowments.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. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Division of Cardiology, 3645 MRL Bldg., UCLA School of Medicine, Los Angeles, CA 90095. Tel.: 310-825-9029; Fax 310-206-5777; E-mail: jweiss{at}ephys.ucla.edu.
1 The abbreviation used is: KACh, muscarinic potassium channels.
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REFERENCES |
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