From the * Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain
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ABSTRACT |
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Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane subunits and cytoplasmic modulatory
subunits. The differential expression and association of
and
subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells,
and their functional expression in heterologous systems provides a tool to study their regulation at a molecular
level. Here, we have studied the effects of Kv
1.2 coexpression on the properties of Shaker and Kv4.2 KV channel
subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kv
1.2
functionally associates with these two
subunits, as well as with the endogenous KV channels of HEK293 cells, to
modulate different properties of the heteromultimers. Kv
1.2 accelerates the rate of inactivation of the Shaker
currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers
sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kv
1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of
the Kv4.2+
channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the
effects of DTT. Finally, hypoxic inhibition of Kv4.2+
currents can be reverted by 70% in the presence of carbon
monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells
and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that
subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability
of Kv4.2+
channels to respond to hypoxia.
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INTRODUCTION |
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Voltage-gated K+ (KV)1 channels help establish the resting membrane potential and modulate the frequency
and duration of the action potentials in excitable cells.
Molecular biology techniques have identified several
mammalian genes encoding the pore-forming subunits of KV channels that can give rise to delayed rectifier or A-type currents upon expression in heterologous systems (Chandy and Gutman, 1995
). The functional and structural diversity of the KV channels'
subunits is further increased by their capacity to form
functional heterotetrameric structures and to associate with modulatory
subunits (for review see Pongs,
1995
; Jan and Jan, 1997
). For example, association of
subunits with some members of the Shaker subfamily results in
heteromultimers with inactivation kinetics
more rapid than those of the corresponding
homomultimers (Rettig et al., 1994
; Chouinard et al., 1995
;
Heinemann et al., 1995
; Majumder et al., 1995
; McCormack et al., 1995
; Morales et al., 1995
), and, even further, some of these
subunits can convert a delayed
rectifier into a rapidly inactivating channel (Rettig et al.,
1994
; England et al., 1995
; Majumder et al., 1995
; Morales et al., 1995
; Heinemann et al., 1996
).
In some tissues, K+ currents exhibit specific properties, such as regulation by oxygen levels (Lopez-Barneo
et al., 1988; Post et al., 1992
; Youngson et al., 1993
). It
has been hypothesized that O2 sensitivity of K+ currents
could be intrinsic to the channels themselves (Ruppersberg et al., 1991
; Duprat et al., 1995
; Weir and Archer,
1995
) or, alternatively, that a membrane-bound O2 sensor or a regulatory subunit of the K+ channels confers
the observed sensitivity (Gonzalez et al., 1992
; Lopez-Barneo, 1994
; Patel et al., 1997
).
In the present work, we have used an heterologous
expression system to study the association of the auxiliary subunit Kv1.2 (formerly Kv
3) with some cloned
KV channels and its possible contribution to the hypoxic sensitivity of the heteromultimers. The KV channels used (Shaker B and Kv4.2) express rapidly inactivating currents comparable to the oxygen-sensitive K+ currents described in some preparations (Lopez-Barneo et
al., 1988
; Gonzalez et al., 1992
). We found subfamily-specific functional interactions between Kv
1.2 and the
different KV channels studied, so that Kv
1.2 coexpression is able to regulate the amplitude of the endogenous HEK293 KV currents, the rate of inactivation of
the Shaker currents, and the redox and oxygen sensitivity of the Kv4.2 currents. The hypoxic response of the
Kv4.2+Kv
1.2 heteromultimers was unaffected by application of reduced glutathione (GSH) in the pipette
solution or in the bath, but was prevented by treatment with DTT (1,4 dithiothreitol) and restored with DTDP
(2,2'-dithiodipyridine), suggesting that reduction of
some, but not all, of the residues susceptible to redox
modulation can disrupt the mechanism underlying the
low pO2 regulation of these channels. Hypoxic inhibition was reverted by carbon monoxide (suggesting the
presence of an hemoproteic O2 sensor in HEK cells)
and remains in excised membrane patches, indicating
that the mechanism of low pO2 inhibition is restricted to the plasma membrane.
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MATERIALS AND METHODS |
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HEK293 Cell Maintenance and Transfections
HEK293 cells were maintained in DMEM supplemented with
10% fetal calf serum (GIBCO BRL), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cells were grown as
a monolayer and plated on squared coverslips (24 × 24 mm)
placed in the bottom of 35-mm Petri dishes at a density of 2-4 × 105 cells/dish the day before transfection. Transient transfections were performed using the calcium-phosphate method (Wigler et
al., 1978) with 1 µg of plasmid DNA encoding the drosophila
Shaker B (H4) K+ channel
subunit (into pRcRSV; Invitrogen
Corp.), or the Kv4.2 K+ channel
subunit (into E42c) alone or
in combination with 2 µg of plasmid DNA encoding the Kv
1.2
subunit into pREP4. In a group of experiments, the cells were
only transfected with 2 µg of Kv
1.2 subunit. In all cases, 0.2 µg
of green fluorescent protein (GFP) in a CMV-promoter expression plasmid (GFPPRK5), was included to permit transfection efficiency estimates (10-40%) and to identify cells for voltage-clamp analysis (Marshall et al., 1995
). Voltage-clamp recordings
revealed typical inactivating currents in 100% of the cells expressing GFP. A group of control cells was obtained by analyzing
the currents present in cells transfected with GFP alone or in untransfected cells. All plasmids used in this study were generously
provided by Drs. E. Marban and G.F. Tomaselli (John Hopkins
University, Baltimore, MD).
Electrophysiological Recordings
K+ currents were studied using either the whole-cell or the outside-out configuration of the patch-clamp technique. The holding potential was 60 or
80 mV, respectively. Isolated HEK cells were studied 1-3 d after transfection. The coverslips with the attached cells were transferred to a small recording chamber (0.2 ml) placed in the stage of an inverted microscope and perfused by gravity with (mM): 141 NaCl, 4.7 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose, 10 HEPES, pH 7.4 with NaOH. The bath solution was connected to ground via a 3 M KCl agar bridge and a Ag-AgCl
electrode. Patch pipettes were double pulled (PP-83; Narishige
Co.) and heat polished (MF-83; Narishige Co.) to resistances
ranging from 1.5-3 M
for whole-cell experiments to 10-15 M
for cell-free recordings when filled with a internal solution containing (mM): 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, 5 MgATP,
pH 7.2 with KOH. Hypoxia was achieved by bubbling the reservoir that fed the perfusion chamber with 100% N2. The final pO2
level in the perfusion chamber was below 10 mmHg. The time
course of the fall in the pO2 was complete within 1 min of solution exchange. In selected experiments, the control solutions
were also bubbled with air to exclude potential artifactual effects
due to the bubbling of the solutions. Whole-cell currents were recorded using an Axopatch 200 patch-clamp amplifier, sampled at
10 and filtered at 2 kHz (
3 dB, four-pole Bessel filter). The series resistance (ranging from 4 to 10 M
) was routinely compensated by 60-80%. Data were leak subtracted on line by a P/4 protocol. K+ currents from macropatches in the outside-out configuration were registered several minutes after excision and were
taken as the difference between the current recorded in a 50-ms
depolarizing pulse to +40 mV from a holding potential of
80
mV and the average current obtained applying four pulses to
+40 mV after inactivating the K+ channels with 200-ms prepulses
to the same potential. To facilitate the subtraction of capacitative
transients, the potential was held at
80 mV during 1 ms between prepulse and pulse. Currents were sampled at 5 and filtered at 1 kHz. Records were digitized with a Digidata-1200 A/D
converter (Axon Instruments), and stored on disk using
PCLAMP version 6.02 software. All the experiments were done at
room temperature (20-22°C).
Data Analysis
Analysis of the data was performed with the CLAMPFIT subroutines of the PCLAMP software and ORIGIN 4.0 software (Microcal Software, Inc.). Pooled data are expressed as mean ± SEM. Statistical comparisons between groups of data were carried out with the two-tailed Student's t test for paired or unpaired data, and values of P < 0.05 were considered statistically significant. The analysis of the differences between two groups of data when comparing more than one variable was done with a fully factorial analysis of variance [(M)ANOVA] using commercial software (SYSTAT; Systat Inc.).
Materials
DTT, DTDP, and GSH were obtained from Sigma Chemical Co. DTT and GSH were prepared fresh and dissolved in the bath or in the pipette solution, and DTDP was first dissolved in ethanol to a concentration of 500 mM, and then diluted in bath solution to a final concentration of 100 µM.
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RESULTS |
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Effects of Kv1.2 on the Amplitude of Shaker, Kv4.2, and
HEK293 Endogenous KV Currents
Untransfected or mock-transfected (GFP alone) HEK293
cells show KV currents of variable size, ranging from 100 to 600 pA at +60 mV. As shown in Fig. 1 A, over the
length of a 100-ms pulse, this endogenous current exhibited almost no inactivation. Outward current was observed at potentials above 20 mV, and peak current typically showed a plateau at potentials more positive than
+40 mV. When HEK293 cells were transfected with
Kv
1.2, there was a significant increase in the current amplitude. The peak current amplitude at +100 mV increased from 0.32 ± 0.07 nA in the mock-transfected cells (n = 7) to 1.38 ± 0.32 in the Kv
1.2-transfected cells
(n = 8). The averaged current-voltage relationships were
statistically different [P = 0.006 with (M)ANOVA], suggesting that Kv
1 subunit could be exerting a chaperon-like effect on the HEK293-endogenous K+ currents.
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Transfection of HEK293 with Shaker or Kv4.2 subunit
cDNA gave rise to large, rapid inactivating currents in all
the cells studied (Fig. 1, B and C). Kv
1.2 coexpression
did not modify significantly the current amplitude, as
shown in the averaged peak current-voltage relationships.
Effects of Kv1.2 on the Kinetics of the Cloned K+ Channels
It has been demonstrated that Kv1.2 is able to modulate the rate of inactivation of some of the members of
the Shaker family when coinjected in Xenopus oocytes
(England et al., 1995
; Majumder et al., 1995
; Morales
et al., 1995
). However, it is not known whether this subunit is able to functionally associate with the K+ channels of the Shal subfamily (Kv4) and modulate their
electrophysiological properties. In our expression system, Kv
1.2 also modulates the kinetics of the recombinant Shaker channels. The traces in Fig. 2 A show that
subunit coexpression accelerates the rate of inactivation and decreased the amplitude of the currents at the
end of a 100-ms pulse. The inactivation time course for
both Shaker and Shaker+
K+ channels was best fitted to
a biexponential function with time constants that exhibited little voltage dependence. The presence of
subunit produced an acceleration of inactivation due
to a significant decrease in the fast time constant at all
the voltages [P = 0.02 with (M)ANOVA] and a less-pronounced decrease in the slow time constant that was not
significant in our analysis (P = 0.054). However, this slow
time constant is very likely distorted by the contribution
of endogenous currents. Fig. 2 B shows a similar analysis
of the kinetics of the Kv4.2 and Kv4.2+
recombinant channels. The time course of inactivation was also fitted
to a biexponential function, but in this case it was not
changed by the presence of
subunit. The same lack of
effect on the activation and inactivation kinetics was observed for the endogenous channels upon association
with
subunit (data not shown).
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Effects of DTT and DTDP on Cloned K+ Channels
The absence of effect of subunit coexpression on the
kinetics of the Kv4.2 channels has two possible explanations: either there is a lack of association between the
two subunits, or, alternatively,
subunit associates with
Kv4.2
subunits to modulate other properties of the
channel. Among these other properties, we have studied their sensitivity to sulfhydryl group reagents. Chemical redox modulation has been demonstrated for different native K+ channels (Weir and Archer, 1995
) as
well as for several cloned K+ channel
subunits (Ruppersberg et al., 1991
; Duprat et al., 1995
). Furthermore, a redox mechanism has been shown to modulate the effects of
subunit on the rate of inactivation of
Kv1.4 K+ channels (Rettig et al., 1994
; Heinemann et al.,
1995
). In our work, the application of the membrane-permeable reducing agent DTT or the oxidizing agent
DTDP to HEK cells expressing Shaker or Shaker+
had
effects similar to those reported in the aforementioned works using other redox agents. Fig. 3 A shows one of
four cells in which application of 100 µM DTDP markedly decreased the rate of inactivation of the Shaker+
currents in an irreversible way, whereas treatment with
2 mM DTT had the opposite effect. As an indicator of
the change in the rate of inactivation, the average modification in the amplitude of the currents at the end of
a 100-ms depolarizing pulse was calculated for three
more cells expressing Shaker+
channels (Fig. 3 A, inset). DTT treatment reduced the amplitude of the current at the end of the pulse by 20.85 ± 7.1%, whereas
DTDP increased this amplitude by 65.5 ± 12.1% (n = 4). The same modifications in the rate of inactivation were observed in cells transfected with Shaker alone
(data not shown). On the contrary, treatment with DTT
or DTDP failed to modify the amplitude or the kinetics
of the currents expressed in cells transfected with Kv4.2
alone (Fig. 3 B, n = 3), in agreement with previous results showing the resistance of channels of the Kv4 subfamily to treatment with other redox agents (Duprat et al., 1995
). When the same experiment was carried out
in Kv4.2+
-transfected cells (Fig. 3 C), we could observe that application of 2 mM DTT produced an irreversible reduction of the current amplitude without any significant change in the kinetics of the currents.
Treatment with DTDP (100 µM) did not modify in a
significant way the amplitude of the currents, but was
able to revert the inhibition induced by DTT. Similar
effects were observed in five more cells expressing Kv4.2+
channels (Fig. 3 C, inset). These results demonstrate that Kv
1.2 is capable of associating with Kv4.2
subunits and, upon coexpression, confers sensitivity
to redox modulation to the Kv4.2+
heteromultimers.
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Effects of Hypoxia on Cloned K+ Channels
Regulation of ion channels by low pO2 was first demonstrated in chemoreceptor cells of the rabbit carotid
body, where an inactivating K+ current was shown to be
reversibly inhibited by hypoxia (Lopez-Barneo et al.,
1988). It is not known whether the oxygen-sensitive K+
channels have an O2 sensing domain or, alternatively,
the oxygen-sensitive cells have other sensor structures
that can affect channel function. To address this question, we have studied the effect of lowering pO2 on the
cloned K+ currents, as well as the possible contribution
of Kv
1.2 to the O2 modulation of the channels. Fig. 4
A shows typical records obtained in depolarizing pulses
to +40 mV from a holding potential of
60 mV for
each channel (endogenous, Shaker, and Kv4.2) alone or
in combination with Kv
1.2. In each case, N2-equilibrated solutions were applied for 2-5 min. Hypoxia did
not modify in a significant way the amplitude or the
time course of the endogenous currents in the mock-transfected (n = 5) or
-transfected (n = 6) cells. The
same lack of effect was observed in all cells expressing
Shaker channels alone (n = 8). In cells transfected with
Shaker+
, hypoxia produced a 10% reduction in the
current amplitude only in one of eight cells studied
(12.5%) and, in Kv4.2-transfected cells, there was a 9%
reduction in the current amplitude in 2 of 13 cells (15%). However, when the effect of hypoxia was studied in Kv4.2+
-cotransfected cells, 38 of 41 cells studied (93%) showed a reduction in the peak current amplitude that ranged from 10 to 40% and averaged 15.48 ± 0.93% at +40 mV (mean ± SEM, n = 38). Even though
in some of the cells low pO2 application seemed to slow
down the time course of inactivation, this effect was not
found to be statistically significant.
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The time course of hypoxic inhibition of the Kv4.2+
cotransfected cells is shown in Fig. 4 B, where the peak
current amplitude at three different voltages is represented against time. The effect of hypoxia was fully
achieved within 1 min after the exchange of the solution and was readily reversible upon washout with the control solution. The hypoxia-induced inhibition of
Kv4.2+
K+ currents was voltage independent (Fig. 4
C), excluding a possible spurious effect due to a shift in
the current-voltage relationship.
Effects of DTT and DTDP on the Response of Kv4.2+
to Hypoxia
The association with Kv1.2 invests Kv4.2 channels with
two new properties, sensitivity to redox modulation and
responsiveness to low pO2 stimulation, making attractive the hypothesis that the redox status of the Kv4.2+
channels could be involved in the effect of hypoxia.
This hypothesis was explored by studying the effect of
hypoxic solutions on these channels after application of reducing or oxidizing agents. Fig. 5 A shows that application of 2 mM DTT produces an irreversible reduction of the current amplitude after which the response
to hypoxia is lost. In these cells the effect of hypoxia
was modified from a 13 ± 1.2% inhibition in control
conditions to a 1.6 ± 0.6% inhibition after DTT treatment. The same protocol was used to study the effect of
the oxidizing agent DTDP (100 µM) on the hypoxic inhibition of the channels (Fig. 5 B). In this case, we can
see that DTDP did not modify the response to hypoxia
of the Kv4.2+
currents (hypoxic inhibition averaged
16.1 ± 2.7% before and 14 ± 1.7% after DTDP treatment, n = 6), although it was able to recover the hypoxic response of cells previously exposed to DTT
(data not shown). These results indicate that the residues of the Kv4.2+
heteromultimers sensitive to DTT
and DTDP treatment are involved in the response of
the channel to acute hypoxia.
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Effects of GSH on Kv4.2+ Currents
Although the previous data suggest that the redox state
could be one of the mechanisms involved in the hypoxic modulation of Kv4.2+ channels, we have explored whether physiological redox modulators such as
GSH have effects comparable to DTT on the currents and on their response to hypoxia. It is noteworthy that
these two reducing agents modify in a similar way the
time course of inactivation of cloned Shaker K+ channels (Ruppersberg et al., 1991
). Additionally, due to its lower membrane permeability, GSH could be helpful
in indicating whether the effects are mainly due to
modification of an intracellular or an extracellular site.
We performed a series of experiments to explore the
effect of pipette application of 5 mM GSH on the amplitude and the kinetics of Kv4.2+
currents and on
their response to low pO2 exposure. Parallel experiments in the same cultures with our normal pipette solution were used as controls. We found that inclusion of
5 mM GSH in the pipette solution did not change the
amplitude of the currents (the peak current at +40 mV
averaged 2.57 ± 0.46 nA in control versus 2.37 ± 0.49 nA in GSH-treated cells, n = 9) nor the inactivation
time course (the two time constants were 11.6 ± 2.9 and 120 ± 6 ms in control cells versus 15 ± 4 and 135 ± 10.2 ms in the presence of GSH, n = 9). When the cells were bathed in a N2-equilibrated solution, all cells in
the two groups showed a reduction of the peak current
amplitude, averaging 16.25 ± 1.6% in control and
16.86 ± 1.83% in GSH-treated cells.
The effects of extracellular application of 5 mM GSH
are shown in Fig. 6, where the peak current amplitude
in depolarizing pulses to +40 mV is plotted against
time. In this cell, application of a N2-equilibrated solution produced the same reduction of the current amplitude before and after bath application of GSH (23 and 22%, respectively). Treatment with 5 mM GSH reversibly decreased the amplitude and rate of inactivation of Kv4.2+ currents, but did not modify the magnitude of the effect of hypoxia. Similar results were observed in four more cells, in which the reduction produced by 5 mM GSH was somehow variable, averaging 33 ± 7%.
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Molecular Mechanism of Low pO2 Inhibition of
Kv4.2+ Currents
The fact that DTT effects could not be reproduced by
treatment with GSH suggests that these two agents are
modifying different thiol groups. To further elucidate
the underlying molecular mechanisms of hypoxic inhibition of Kv4.2+ channels, we have performed some experiments in excised membrane patches, devoid of
potential intracellular mediators. Fig. 7 shows the effect
of hypoxia on the peak current amplitude recorded
with the protocol described in MATERIALS AND METHODS in an outside-out patch obtained from a Kv4.2+
-transfected cell. The current traces corresponding to
the numbers in the plot are also shown in Fig. 7. As in
the whole-cell experiments, perfusion with N2-equilibrated solution produced a reduction in the amplitude of the current that reverted upon washout with normoxic solution. The same inhibition was observed in
five more patches, with an average effect of 22.0 ± 2.5%. The average peak currents in these six patches
was 106.3 ± 31.0 pA.
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These data indicate that acute hypoxia is acting
through a membrane-delimited pathway to produce
the inhibition of Kv4.2+ currents, suggesting that either the Kv4.2+
channel proteins are intrinsically oxygen sensitive or, alternatively, there is a closely associated but distinct oxygen-sensing element that is endogenously expressed in the membrane of the host cell.
The available evidence argues in favor of this latter possibility in several oxygen-sensitive tissues (Gonzalez et al.,
1992
), as well as in heterologous expression systems
such as COS cells (Patel et al., 1997
) and HEK293 cells
(Fearon et al., 1999
). To explore this possibility, we
have studied the effect of carbon monoxide on the hypoxic response of Kv4.2+
. CO is a very inert gas that in biological systems only reacts with hemoproteins.
Fig. 8 shows the effect of CO on the inhibition of
Kv4.2+
currents by low pO2. Fig. 8 shows the peak
current amplitudes at two different voltages obtained
in a cell while perfusing with control solution (pO2 = 150 mmHg), with a hypoxic solution equilibrated with
100% N2 (pO2 < 10 mmHg), with a hypoxic solution
equilibrated with a gas mixture containing 20% CO in
N2 (pO2 < 10 mmHg, estimated pCO = 150 mmHg),
and after returning to the control solution. We found
that CO is reverting in a significant extent the inhibition observed with hypoxia with a time course even
faster that the onset of hypoxic application, indicating
that CO is able to successfully replace O2 at the O2-sensing molecule, albeit with smaller affinity. The same reversion was observed in 10 more cells studied, in which CO prevented or reversed by 69.5 ± 3.2% the low pO2-induced inhibition of Kv4.2+
currents, so that the average hypoxic inhibition decreased from 16.2 ± 1.4%
to 5.04 ± 0.9% (Fig. 8 B).
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DISCUSSION |
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The expression of recombinant channels in heterologous systems has proved a useful tool to characterize
ionic channels in isolation. Transient transfection of
HEK293 cells provides an efficient expression of channel proteins in a mammalian cell background, and offers an additional advantage for the present work, due
to the absence of endogenous Kv subunits in HEK293
cells (Uebele et al., 1996
). The presence of endogenous
subunits did not represent a problem due to the
clear differences, both in amplitude and kinetics, between these endogenous currents and the currents
through transfected KV channels (see Fig. 1). The significant increase in the endogenous current amplitude
obtained in Kv
1.2-transfected cells suggest that this
subunit interacts with the endogenous
subunits. A
chaperon-like effect has been reported for several
subunits acting on specific KV channel
subunits (Chouinard et al., 1995
; Fink et al., 1996
; Shi et al., 1996
).
Studies on subunit-mediated effects on K+ channels have been primarily focused on the modifications
induced in the inactivation kinetics of the heteromultimers. These studies indicate that several
subunits
(Kv
1, Kv
3, and the Drosophila homologue Hk) are
able to increase the rate of inactivation of specific
members of the Shaker subfamily that express A-type
currents or convert delayed-rectifier currents into A-type
currents (see INTRODUCTION). This interaction between Kv
1 and members of the Shaker subfamily has
been confirmed with immunohistochemical studies
that show the association and colocalization of these
-
complexes (Rhodes et al., 1995
, 1997
; Nakahira et
al., 1996
; Yu et al., 1996
). It has also been reported the
existence of selective interaction between both Kv
1
and Kv
2 and the mammalian Shal homologue Kv4.2
(Nakahira et al., 1996
), but functional analysis has failed
to reveal a change in the inactivation properties of the
members of the Shal subfamily when coexpressed with
Kv
1 subunit (Heinemann et al., 1996
; Yu et al., 1996
).
In agreement with these reports, we found that coexpression with Kv1.2 produces a significant change in
the rate of inactivation of the Shaker channels due to a
decrease in the fast time constant. Besides, the fact that
the acceleration of the channel inactivation by Kv
1.2
does not reduce the peak current amplitude (Fig. 1 B) suggests that Kv
1.2 is also increasing the surface expression of Shaker channels. Also in agreement with
previous data, we found no changes in the inactivation
rate of the Kv4.2 currents upon Kv
1.2 coexpression.
However, the association is functionally assessed by the
acquisition by the Kv4.2+
currents of new property, namely the sensitivity to sulfhydryl group reagents (Fig.
3). Another proof of this functional association of
Kv4.2 with Kv
1.2 is the capability of Kv4.2+
currents
to respond to low pO2. This response was only consistently observed in our expression system with this particular
+
subunit combination, and consisted in a reversible reduction of the current amplitude upon exposure to hypoxic solutions (Figs. 4-8). One important
aspect to consider regarding this effect is whether we
are dealing with a metabolic or an allosteric-type mechanism. Given the speed of the effect of hypoxia, and
the presence of 5 mM ATP in the intracellular solution,
a direct action of hypoxia seems more likely than a response to altered cellular metabolism. Actually, hypoxic
inhibition of native A-type K+ channels has been slow
to occur in excised membrane patches (Ganfornina and Lopez-Barneo, 1991
), and the data presented in
Fig. 7, showing the same effect of hypoxia in excised
patches in the absence of potential intracellular mediators, strongly suggest that the response to hypoxia is a
membrane-delimited mechanism. In addition, the persistence of the low pO2 inhibition in a cell-free preparation confirms that Kv4.2 is able to coassemble with
Kv
1.2.
The modifications in the hypoxic response after application of freely membrane-permeable oxidizing and
reducing agents suggest that hypoxic sensitivity can be
modulated by the redox status of the channel proteins
and that the same cysteine residues modified by DTT
and DTDP are involved in the low pO2 regulation of the Kv4.2+ channels. However, the absence of effect of
GSH when applied intracellularly argues against a role
for redox modulation under physiological conditions,
and also excludes the possibility that the effect of low
pO2 on the Kv4.2+
channels could be attributable to
the redox status of the cytoplasmic
subunits. On the
other hand, extracellular GSH application does not interfere with the hypoxic response of the channel, supporting the idea that hypoxia and reducing agents can
inhibit Kv4.2+
currents through different mechanisms. The fact that Shaker and Shaker+
channels are
also modified by these agents, but insensitive to hypoxia, stresses out the fact that the effect of low pO2 as
a physiological stimulus is not simply achieved by the
reduction of a sulfhydryl group. Redox modulation of
Shaker or Shaker+
currents was able to change their
rate of inactivation, but none of these maneuvers rendered the channels sensitive to hypoxia (data not
shown). Furthermore, in contrast with DTT effect, application of hypoxic solutions did not modify the rate
of inactivation of the channels (see Fig. 4 A). These observations indicate that O2 sensing must have some specific structural requirements that seem to be achieved
in our expression system by the combination of Kv4.2
subunits with Kv
1.2 subunits. With respect to the molecular nature of the O2-sensing mechanism, there are
two possibilities: first, the Kv4.2+
channels themselves
are the O2-sensing devices and, second, there is some other O2-sensing molecule endogenously present in
HEK293 cells (Wang and Semenza, 1993
; Fearon et al.,
1999
) capable of interacting with Kv4.2
subunits only
when a
subunit is also present. Data on the literature
showing that other structurally distinct channels are
also O2 sensitive in this cell line (Fearon et al., 1997
; McKenna et al., 1998
) support the second possibility,
and data on the present study locate this O2 sensor in
the plasma membrane. Since the only known targets of
CO in biological systems are reduced hemoproteins
with accessible iron sites, our observation that CO is
able to interact with this putative O2 sensor, replacing O2 and preventing the inhibition of K+ currents (Fig.
8), strongly suggests that the intrinsic O2 sensor of
HEK293 cells is a hemoprotein.
The physiological relevance of the findings reported
here is difficult to evaluate because neither the molecular nature of the O2-sensitive K+ channels nor the distribution and coexpression of Kv4.2 with Kv1 in native
tissues are known. However, our results showing evidence that
subunits provide hypoxic sensitivity to specific KV channel
subunits put forth the interesting
possibility of the existence of tissue-specific modulatory
subunit(s) that confer hypoxic sensitivity to the expressing tissues. This idea is supported by a recent report by Patel et al. (1997)
in which a new K channel subunit, Kv9.3, that does not form a channel itself, is
able to coassemble with Kv1.2 and increase the probability of the heteromultimeric channels to be modulated by hypoxia. Finally, our findings provide new
clues in the search for the molecular mechanisms of O2
sensing in hypoxia-sensitive tissues, raising a completely
new set of questions requiring further investigation.
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FOOTNOTES |
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Address correspondence to Constancio González, Departamento de Fisiología, Facultad de Medicina, c/ Ramón y Cajal s/n, 47005 Valladolid, Spain. Fax: 34 83 423588; E-mail: constanc{at}ibgm.uva.es
Original version received 18 December 1998 and accepted version received 31 March 1999.
Portions of this work have previously appeared in abstract form (Pérez-García, M.T., J.R. López-López, and C. Gonzalez. 1998. J. Physiol. 509P:36P).We thank Drs. E. Marban and G.F. Tomaselli for kindly providing the plasmids used in this study, and M. de los Llanos Bravo for technical help.
This study was supported by Spanish Dirección General de Investigación Científica y Técnica grant PB97/0400 to C. González.
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Abbreviations used in this paper |
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GFP, green fluorescent protein; GSH, reduced glutathione; KV channel, voltage-gated K+ channel; (M)ANOVA, fully factorial analysis of variance.
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