1Departments of Physiology and Biophysics and 2Rammelkamp Center for Education and Research, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970
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ABSTRACT |
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Overholt, Jeffrey L.,
Eckhard Ficker,
Tianen Yang,
Hashim Shams,
Gary R. Bright, and
Nanduri R. Prabhakar.
HERG-Like Potassium Current Regulates the Resting Membrane
Potential in Glomus Cells of the Rabbit Carotid Body.
J. Neurophysiol. 83: 1150-1157, 2000.
Direct evidence for a
specific K+ channel underlying the resting membrane
potential in glomus cells of the carotid body has been absent. The
product of the human ether-a-go-go-related gene (HERG) produces inward
rectifier currents that are known to contribute to the resting membrane
potential in other neuronal cells. The goal of the present study was to
determine whether carotid body glomus cells express HERG-like
K+ current, and if so, to determine whether a HERG-like
current regulates the resting membrane potential. Freshly dissociated rabbit glomus cells under whole cell voltage clamp exhibited slowly decaying outward currents that activated 20-30 mV positive to the
resting membrane potential. Raising extracellular K+
revealed a slowly deactivating inward tail current indicative of
HERG-like K+ current. HERG-like currents were not found in
cells resembling type II cells. The HERG-like current was blocked by
dofetilide (DOF) in a concentration-dependent manner
(IC50 = 13 ± 4 nM, mean ± SE) and
high concentrations of Ba2+ (1 and 10 mM). The biophysical
and pharmacological characteristics of this inward tail current suggest
that it is conducted by a HERG-like channel. The steady-state
activation properties of the HERG-like current
(Vh = 44 ± 2 mV) suggest that
it is active at the resting membrane potential in glomus cells. In
whole cell, current-clamped glomus cells (average resting membrane
potential,
48 ± 4 mV), DOF, but not tetraethylammonium, caused
a significant (13 mV) depolarizing shift in the resting membrane
potential. Using fluorescence imaging, DOF increased
[Ca2+]i in isolated glomus cells. In an
in-vitro carotid body preparation, DOF increased basal sensory
discharge in the carotid sinus nerve in a concentration-dependent
manner. These results demonstrate that glomus cells express a HERG-like
current that is active at, and responsible for controlling the resting
membrane potential.
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INTRODUCTION |
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Carotid bodies are sensory organs that regulate
respiratory responses to alterations in arterial blood oxygen.
Hypoxemia (low arterial O2) augments the sensory
discharge of the carotid bodies, and the sensory information is
conveyed via the carotid sinus nerve to respiratory neurons in the
brain stem. Reflexes arising from the carotid bodies are important for
maintaining homeostasis during hypoxemia that occur in many
physiological situations including sojourn at high altitude, and in
pathophysiological conditions such as sudden infant death syndrome
(SIDS) (Perrin et al. 1984). Morphologically the carotid
body is composed of neurotransmitter-enriched glomus (type I) cells and
glial-like type II cells. There is much evidence that the glomus cells
are the initial sites of sensory transduction. It has been proposed
that hypoxia causes membrane depolarization in glomus cells causing
influx of Ca2+ through voltage-gated
Ca2+ channels, leading to release of
neurotransmitter(s) that act on apposing afferent nerve terminals to
increase sensory discharge in the carotid sinus nerve (for reviews see
Fidone et al. 1990
; Gonzalez et al. 1994
;
Prabhakar 1994
).
The cellular basis for the initial depolarization of glomus cells
during hypoxia remains poorly understood. Several studies have reported
that hypoxia inhibits outward K+ currents in
glomus cells. Therefore it was proposed that the hypoxia-sensitive
K+ channels contribute to the initial
depolarization that is essential for the transduction of the hypoxic
stimulus (Lopez Barneo et al. 1988; Wyatt et al.
1995
). However, recent studies have questioned whether
inhibition of these K+ channels is central to the
transduction process at the carotid body. First, these channels are not
active at the reported resting membrane potentials of glomus cells
(Gonzalez et al. 1994
). Second, known blockers of these
K+ channels had no affect on basal or hypoxia
stimulated sensory activity of the carotid body or on intracellular
Ca2+
([Ca2+]i) in isolated
glomus cells (Buckler 1997
; Cheng and Donnelly 1995
; Lahiri et al. 1998
; however, see
Wyatt et al. 1995
). More recently, Buckler
(1997)
identified a K+-selective
"leak" conductance that is sensitive to hypoxia. However, its role
in transduction remains elusive, because there is no known selective
pharmacological blocker of this conductance. Consequently, which
K+ current regulates the resting membrane
potential and the role of K+ channels in the
transduction process of the hypoxic stimulus remain uncertain. It
follows that identification of specific channel(s) active at the
resting membrane potential is of seminal importance to understand the
basis for hypoxic depolarization in glomus cells.
Inwardly rectifying K+ channels contribute to the
resting membrane potential in many different cell types. One such
channel is the protein encoded by the human ether-a-go-go-related gene (HERG). HERG K+ channels were originally
identified as molecular targets for mutations underlying one form of
the long QT syndrome, a genetic disease with delayed cardiac
repolarization (Curran et al. 1995). The main features
of HERG current are a peculiar inward rectification mechanism and its
unique sensitivity to methanesulfonanilide drugs such as E-4031 and
dofetilide (Ficker et al. 1998
; Jurkiewicz and
Sanguinetti 1993
; Smith et al. 1996
;
Snyders and Chaudhary 1996
; Trudeau et al.
1995
). HERG-like channels have also been identified in cells of
neural crest origin, such as neuroblastoma cell lines, PC12 cells, and
quail neural crest cells, where they regulate the resting membrane
potential (Arcangeli et al. 1995
, 1997
;
Bianchi et al. 1998
; Shi et al. 1997
).
The goal of the present study was to determine whether carotid body
glomus cells express HERG-like K+ current, and if so, to
determine whether HERG-like currents regulate the resting membrane
potential. However, identification of HERG-like currents in glomus
cells is difficult due to the presence of a large outward
K+ current. Therefore we took advantage of the unique
characteristics of HERG channels to isolate the HERG-like current. If
HERG current is present, stepping to hyperpolarized potentials from a
holding potential of 0 mV (to inactivate the outward K+
current) should give rise to a rapidly activating (actually removal of
inactivation) inward K+ current that decays in a time- and
voltage-dependent manner as the channels close. This process forms a
"nose" in the tail current that is characteristic of HERG current
and should be blocked by dofetilide.
Based on the criteria above, our results show that glomus cells of the
rabbit carotid body express HERG-like K+ current. Most
importantly, the characteristics of this HERG-like current suggest that
it is active at the resting membrane potential. Furthermore, block of
HERG-like current depolarizes glomus cells, increases
[Ca2+]i, and augments sensory activity under
normoxia in an in-vitro carotid body preparation. This study is the
first to show the molecular basis for a K+ current active
around the resting membrane potential that is directly involved in
controlling the resting membrane potential in glomus cells of the
carotid body. Preliminary results from this study have been reported
previously (Overholt et al. 1999).
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METHODS |
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Isolation of rabbit carotid body cells
Carotid body cells were acutely isolated from adult male rabbits
as described previously (Overholt and Prabhakar 1997).
Dissociated cells were maintained at 37°C in a
CO2 incubator in medium composed of a 50/50
mixture of Dulbecco's modified Eagle's medium (DMEM) and HAM F12
(GIBCO) supplemented with antibiotics (penicillin and streptomycin),
10% fetal bovine serum, and insulin, transferrin, and selenium (ITS,
Sigma). Experiments were performed at room temperature. Cells were used
within 4-36 h.
Membrane current recording
K+ currents were measured in the whole
cell configuration of the patch-clamp technique (Hamill et al.
1981) using an Axopatch 200 amplifier (Axon Instruments). Patch
pipettes had resistances of 2-5 M
when filled with (in mM) 100 K
aspartate, 20 KCl, 2 MgCl2, 1 CaCl2, 10 EGTA, and 10 HEPES, pH 7.2, adjusted to
300 mosM with glucose. The standard extracellular solution was composed of (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, and 10 glucose, pH 7.4. The
K+ concentration in the extracellular solution
was varied by equimolar replacement of NaCl with KCl. Unless indicated,
current recordings were not corrected for leak. Current traces were
sampled at 2.4 kHz and filtered at 1 kHz for off-line analysis. In some
recordings, series resistance errors were compensated to at least 70%.
PClamp programs (Axon Instruments) were used for data acquisition and analysis. The peak of the tail currents was determined by extrapolation of the deactivating tail current to the beginning of the voltage step.
The extracellular solution was changed using a fast-flow device
consisting of a linear array of borosilicate glass capillary tubings
(Overholt and Prabhakar 1997
).
Membrane potential recording
Membrane potential measurements were made using an Axopatch 200 amplifier in the current-clamp mode. The intracellular solution had the
following composition (in mM) 120 K glutamate, 20 KCl, 5 Mg-ATP, 5 EGTA, 5 HEPES, and 0.1 Tris-GTP, pH 7.2. Cells were perfused with
Krebs-Heinseliet buffer equilibrated with 5% CO2 in air (in mM): 120 NaCl, 4.8 KCl, 1.5 CaCl2, 2.2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose, pH 7.4. Drugs were added
to the extracellular perfusate. Input resistance was measured from
hyperpolarizing current injections of 10 pA applied from the resting
membrane potential.
Measurements of [Ca2+]i
Changes in [Ca2+]i
in individual glomus cells were measured as described previously
(Bright et al. 1996). Briefly, cells were plated on
glass coverslips and preincubated in 3 ml of serum free DMEM medium
containing 5 µM Indo-1-PE3 (Texas Fluorescence Lab) for 60 min at 37 °C. Subsequently, they were placed in a gas-tight, temperature
regulated chamber (Bioptics) and superfused with Hank's Balanced Salt
Solution (HBSS) equilibrated with 21% oxygen and 5%
CO2. Images were recorded with a Zeiss LSM-410
equipped with a UV laser. Excitation was 360 nm with emission at 408 and 475 nm. Data are expressed as ratio values due to unstable
Rmin and Rmax values within the cell using
either ionomycin or 8Br-A23187. Cells responding to hypoxia with
increases in [Ca2+]i were
considered to be glomus cells.
Recording of carotid body sensory discharge in vitro
The method for recording sensory discharge from isolated carotid
bodies has been described previously (Prabhakar et al.
1995). Briefly, the carotid bifurcation along with the carotid
sinus nerve was excised from anesthetized, adult rabbits. The carotid bifurcation was placed in a recording chamber, and the common carotid
artery was cannulated and perfused with DMEM solution (pH 7.3;
temperature, 36 ± 1°C) at a rate of 3.5 ± 0.5 (SE) ml/min. Chemoreceptor activity was recorded from the sinus
nerve using platinum-iridium electrodes. Sensory discharge frequency is
expressed as impulses/second. Hypoxia (PO2 = 38 ± 6 mmHg for 1-2 min) augmented the sensory activity, suggesting that
the action potentials were of chemoreceptor origin. The superfusion
medium was equilibrated with room air (normoxia). The partial pressure
of oxygen (pO2) in the medium was measured using a blood
gas analyzer.
Drugs
DofetilideN-[4-(-{-[4-(methanesulfonamino)-phenoxyl]-ethylethylamino}ethyl)phenyl] methansulfonamide was a gift from Pfizer Central Research. All other chemicals were purchased from Sigma.
Data analysis
Statistical analysis was evaluated by a paired or unpaired t-test, or one-way ANOVA combined with Tukey's test, where appropriate. P values <0.05 were considered significant. Summary data are expressed as means ± SE.
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RESULTS |
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Glomus cells of the rabbit carotid body express a HERG-like K+ current
Dissociated cells from carotid bodies contain both glomus
and type II cells. Glomus, but not type II cells, express
Na+ and large, outward, delayed rectifier-like
K+ currents (Urena et al. 1989).
Therefore we used the presence of large, outward
K+ currents and inward Na+
current to distinguish glomus cells from type II cells
(Overholt and Prabhakar 1997
). Figure
1A shows typical currents
elicited by 75-ms depolarizing voltage-clamp steps from a glomus cell
held at
85 mV measured in an extracellular solution containing 5 mM K+. Rapidly inactivating, inward
Na+ currents followed by large, outward
K+ currents typical of glomus cells can be seen.
Figure 1B shows the outward K+
currents elicited by longer (2.8 ms) depolarizing voltage-clamp steps
(note, the Na+ current cannot be resolved on this
time scale). Outward currents were slowly decaying and could be
described best by double exponential functions (see legend). Figure
1C shows the activation and inactivation properties of the
outward K+ current. Activation properties were
determined by analyzing peak current amplitudes using protocols shown
in Fig. 1B. The threshold for activation was reached between
40 and
30 mV and was half-maximal at 4.7 mV. To determine
steady-state inactivation, cells were held at +20 mV and stepped for
1 s from +20 to
130 mV in 10-mV increments. Peak currents were
analyzed on return to +20 mV. Steady-state inactivation could be fitted
by a Boltzman equation with Vh at
73
mV. The characteristics of the outward K+
currents are similar to those described in rabbit glomus cells by other
investigators (Lopez-Lopez et al. 1993
) and suggest that they would not be active at the resting membrane potential in glomus
cells.
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Once establishing that a recording was from a glomus cell, we
tested whether the same cells also express HERG-like
K+ current. HERG-like currents cannot be readily
identified under the conditions in Fig. 1B, due to rapid
inactivation at depolarized potentials, but can be substantially
amplified at more hyperpolarized potentials by raising the
concentration of extracellular K+
([K+]o). Figure
1D shows an example of currents elicited by the same protocol and in the same cell shown in Fig. 1B, but the
extracellular solution was changed to one containing 70 mM
K+ by equimolar replacement of NaCl by KCl.
Elevating [K+]o did not
change any of the kinetic parameters found in normal K+. The holding current measured at 85 mV
showed only a minor increase from
12.8 ± 1.8 to
23.8 ± 2.1 pA (n = 7) in 5 and 70 mM
[K+]o, respectively. This
small change in current amplitudes could not be prevented by 50 µM
Ba2+, which should block current conducted by
classical inward rectifier K+ channels in the
KIR family. Rather, Fig. 1D shows that
the most dramatic change observed in current recordings performed in
elevated [K+]o was the
appearance of a slowly deactivating tail current component (arrow),
indicative of inward rectifying K+ current.
Figure 2 further characterizes this
inward rectifier K+ current in glomus cells.
Figure 2A shows that this current activates very slowly.
After a 50-ms depolarization, tail currents elicited on return to 85
mV were instantaneous and decayed with a time constant of 22.1 ± 2.3 ms. More importantly, after a 3-s depolarization, a slow component
appeared that showed delayed onset and decayed with a much slower time
constant of 157.7 ± 1.4 ms (n = 3). The slow
development of this component suggests that the current activates slowly (i.e., seconds, not milliseconds) as is characteristic for HERG
channels. This "nose" that develops over time in tail currents
recorded in elevated
[K+]o indicates that the
inward rectifier K+ current is conducted by a
HERG-like K+ channel. Figure 2B shows
current traces elicited by a 200-ms step from a holding potential of 0 to
120 mV in a glomus cell. This protocol is optimal for observing
the HERG-like current in glomus cells, because HERG channels are
maximally activated at 0 mV and the large, outward
K+ currents are inactivated. It can be seen
that increasing the extracellular K+
concentration from 5 to 40 mM clearly exposes the HERG-like tail current and suggests that it conducts K+. Figure
2C shows a family of inward tail currents elicited by hyperpolarizing steps from 0 mV. HERG-like tail currents first increased for a few milliseconds, due to rapid removal of inactivation, and then deactivated in a time- and voltage-dependent manner. The delayed rectifier-like outward currents can also be seen on return to 0 mV due to removal of inactivation during the
hyperpolarizing steps. These results show that the inward rectifier
current in glomus cells conducts K+ (increase in
conductance in response to increase K+), requires
hundreds of milliseconds to activate (slow development of the tail
current), and deactivates in a time- and voltage-dependent manner
(channel closing). These are all characteristic of currents conducted
by HERG-like K+ channels identified in other
cells (Bianchi et al. 1998
). In contrast, type II cells,
identified by characteristic small outward currents did not exhibit
these HERG-like currents when tested under identical conditions
(n = 4, data not shown).
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Pharmacological characterization of HERG-like currents in glomus cells
Heterologously expressed HERG channels are blocked with highest
affinity and selectivity by class III antiarrhythmic
methanesulfonanilide drugs such as dofetilide (Arcangeli et al.
1997). To further confirm that HERG channels underlie the
HERG-like current in glomus cells, we tested the effect of dofetilide
on this current. Figure 3A shows characteristic HERG-like tail currents elicited on
hyperpolarization from 0 to
120 mV before and during exposure to 0.01 or 1 µM dofetilide in the extracellular solution. It can be seen that
HERG-like K+ currents in glomus cells were
blocked by dofetilide in a concentration-dependent manner. However, the
effects of micromolar concentrations of dofetilide were not reversible.
The effect of dofetilide on the HERG-like tail current is further
characterized in Fig. 3B, which shows the relative effect of
a range of concentrations of dofetilide on the current. From these
experiments, we determined the IC50 of dofetilide
block to be 12.7 ± 4.2 nM (n = 11). In marked
contrast, transient outward currents were not affected (1 µM
dofetilide actually increased the outward current elicited on return to
0 mV by 7.1 ± 6.0%, see Fig. 3A; n = 8).
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Cations such as Ba2+ have been used to
differentiate HERG-like inward currents elicited in elevated
[K+]o from classical
inward rectifier currents. HERG-like K+ currents,
as well as heterologously expressed HERG channels, proved to be less
sensitive to block by external Ba2+ than inward
rectifiers in the Kir gene family (Kubo et
al. 1993). In marked contrast to the strong block of classical
inward rectifiers by 50 µM Ba2+, Fig.
3C shows that the HERG-like current in glomus cells is little affected by 50 µM Ba2+ (average
reduction 7.5 ± 1.1% at
120 mV; n = 5). Figure
3C also shows that the HERG-like current is reduced by high
(mM) concentrations of Ba2+. One and 10 mM
Ba2+ reduced HERG-like currents at
120 mV by
34.1 ± 1.7 (n = 5) and 61.7 ± 4.2%
(n = 4), respectively. Outward currents at 0 mV were only moderately reduced. Because TEA has been shown to block outward K+ currents in glomus cells, we tested the
effects of TEA on the HERG-like current. Figure 3D shows
that 10 mM TEA blocked a much larger proportion of the outward current
elicited on return to 0 mV (77.3 ± 4.1%; n = 8)
than of the HERG-like current elicited at
120 mV (35.9 ± 2.5%;
n = 8). These results demonstrate that the
pharmacological profile of the HERG-like current in glomus cells is
similar to that described for HERG currents in other cells
(Bianchi et al. 1998
), and further support the idea that inward rectifier K+ current in glomus cells is
conducted by HERG-like channels.
HERG-like K+ current regulates resting membrane potential in glomus cells
We next wanted to elucidate the functional role of the HERG-like
current in glomus cells. To determine whether the HERG-like current is
active at the resting membrane potential, we first examined the
steady-state activation properties of this current. Figure
4A shows a family of current
traces recorded on return to 100 mV after a range of 27.6 s test
potentials from a glomus cell exposed to an extracellular solution
containing 70 mM K+. The results of these
experiments are summarized in Fig. 4B, which shows
normalized current measured on return to
100 mV. It can be seen that
the data are well fit by a Boltzman equation with
Vh at
44.0 ± 2.1 mV and slope
conductance k of
10.5 ± 2.1 (n = 7).
The steady-state activation properties suggest that the HERG-like
K+ current in glomus cells is active around
50
mV, which is close to the resting membrane potential of these cells
(see Table 1).
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To assess whether HERG-like current could regulate the resting membrane
potential in glomus cells, we examined the effect of dofetilide on the
resting membrane potential. For these current-clamp experiments we used
nanomolar concentrations to demonstrate the reversibility of dofetilide
effects. In addition, a bicarbonate buffered extracellular solution was
used. Stable measurements of the resting membrane potential were
recorded in 44 glomus cells current clamped in the whole cell
configuration. Only five of these cells displayed spontaneous action
potentials, which diminished over time during whole cell dialysis. On
average, the resting membrane potential was 48.3 ± 1.9 mV
(range
67.2 to
33.2 mV, n = 32), and the input
resistance was 3.0 ± 0.3 G
(n = 12). Most importantly, 150 nM dofetilide caused a significant and reversible depolarization of
13 mV. In contrast, 10 mM TEA, which completely blocked the large, outward current, had no effect on the resting membrane potential. The results from these experiments are summarized in Table 1. These results show that the HERG-like current is involved
in regulating the resting membrane potential in glomus cells.
Dofetilide increases [Ca2+]i in glomus cells
A depolarization-induced influx of Ca2+ through membrane Ca2+ channels is an essential step in chemotransduction at the carotid body. Therefore if the HERG-like current is active at the resting membrane potential, then inhibition of this current should cause depolarization and elevate [Ca2+]i in glomus cells. To test this possibility, we monitored the effect of dofetilide on [Ca2+]i in glomus cells using Indo-1-PE3, a calcium-sensitive fluorescent dye. As a control, we also tested the effect of tetraethylammonium (TEA) on [Ca2+]i. Figure 4C shows the effect of 10 mM TEA and 1 µM dofetilide on [Ca2+]i in a representative glomus cell. It can be seen that TEA had no effect on [Ca2+]i, whereas the same cell responded with a prompt increase in [Ca2+]i in response to dofetilide. [Ca2+]i returned slowly to baseline levels after wash out of dofetilide. Of the 43 cells tested, 33 responded with an increase in [Ca2+]i in response to dofetilide (change in ratio from 1.2 ± 0.02 to 2.0 ± 0.03), similar to that seen with the control response to hypoxia (PO2 = 32 ± 4 mmHg; change in ratio from 1.2 ± 0.02 to 1.8 ± 0.02). In contrast, TEA caused an increase in [Ca2+]i in only one of these cells (Fig. 4D). These results suggest that the HERG-like current plays a functional role in glomus cells at the cellular level.
Dofetilide increases baseline sensory discharge in the in-vitro carotid body
To determine whether the functional aspects of the HERG-like current at the cellular level translate to the organ, we monitored the effects of dofetilide on sensory discharge of isolated carotid bodies using an in-vitro preparation. The in- vitro carotid body preparation avoids potential systemic effects of dofetilide on the cardiovascular system that may influence chemosensory activity. Figure 5A shows the effects of a range of concentrations of dofetilide on baseline sensory discharge from the carotid sinus nerve in a representative preparation. As can be seen, dofetilide increased the sensory discharge in a concentration-dependent manner. As little as 0.3 µM dofetilide significantly enhanced the sensory activity, and at 3 µM, baseline activity was increased by 98 ± 6% (P < 0.01; n = 8). These results are summarized in Fig. 5B. The effects of dofetilide on baseline carotid body activity were first discernable between 3 and 5 min, had plateaued by 10 min, and did not return to control levels within 15 min after termination due to the high concentrations used. These results demonstrate that block of HERG channels mimics the effects of hypoxia on the sensory discharge of the carotid body and establish the functional significance of the HERG-like current at the organ level.
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DISCUSSION |
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HERG channels are members of the voltage-gated ether-a-go-go
K+ channel family and are characterized by an
unusually slow current activation and deactivation, paired with a fast
C-type inactivation mechanism. In the present study, we clearly
identify a HERG-like current component in glomus cells using
electrophysiological and pharmacological methods. We found HERG-like
tail currents after long depolarizing steps under high
[K+]o only in those cells
that displayed the large, outward K+ current,
characteristic of glomus cells (Urena et al. 1989). HERG-like current was not found in cells displaying only small outward
K+ currents characteristic of type II cells,
suggesting that expression of the HERG-like current in the carotid body
is confined to glomus cells. Whether nerve and/or vascular tissue in
the carotid body also express HERG-like current remains to be
established. The HERG-like tail current in glomus cells increased for a
few milliseconds before deactivating at hyperpolarized membrane
potentials, a characteristic unique to HERG channels. This "nose"
clearly identifies this current as carried by HERG-like gene products
because it results from a unique inactivation mechanism that recovers
rapidly at negative potentials. The kinetics of the currents
(especially Fig. 2C) are identical to HERG-like currents
identified by electrophysiological and molecular means in other cell
lines (Bianchi et al. 1998
). Further, the HERG-like
current is carried by K+ ions (Fig.
2B), shows time- and voltage-dependent deactivation (Fig.
2C), slow current activation (Fig. 2A), and
steady-state activation (Fig. 4B), all characteristics of
HERG channel currents. However, it was not possible to characterize
HERG-like outward currents in glomus cells because of the much larger
outward K+ currents and the rapid inactivation
process at depolarized potentials. This precludes a quantitative
analysis of the time course for activation of the HERG-like current.
The kinetic evidence that a HERG channel protein conducts the HERG-like
current in glomus cells is further corroborated by pharmacological
evidence. In contrast to conventional inward rectifier K+ channels (Kubo et al. 1993),
the HERG-like currents were little affected by micromolar
concentrations of Ba2+. However, they were
inhibited by mM concentrations of Ba2+ as
reported for HERG-like channels studied in neuroblastoma and microglial
cells (Arcangeli et al. 1995
; Zhou et al.
1998
). Furthermore, inward tails were blocked with nanomolar
affinity by dofetilide, a potent and specific blocker of HERG
K+ channels (Ficker et al. 1998
;
Snyders and Chaudhary 1996
). Consistent with previous
reports we also found that the effects of dofetilide were only
partially reversible at concentrations higher than 1 µM
(Ficker et al. 1998
). These results show that a HERG
channel protein conducts the HERG-like current in glomus cells. HERG
currents arise from expression of three closely related HERG genes,
ERG1-3, which are widely expressed in nervous tissue (Shi et
al. 1997
; Warmke and Ganetzky 1994
). However,
which of the specific HERG gene(s) are expressed in glomus cells remain
to be investigated.
Several observations in the present study show that the HERG-like
K+ current plays a functional role in regulating
the resting membrane potential in rabbit glomus cells. Activation of
the HERG-like K+ current was half-maximal at 44
mV, and the threshold for current activation was reached between
70
and
60 mV (Fig. 4B). This is sufficiently negative to
stabilize the membrane potential between
65 and
40 mV, as measured
in current-clamp recording from glomus cells (Table 1). On the other
hand, it is not expected that the O2-sensitive,
outward K+ currents would be open at the resting
membrane potential (Lopez-Lopez et al. 1993
;
Wyatt et al. 1995
) (also Fig. 1C).
Furthermore, TEA, which blocks the O2-sensitive
K+ currents, neither depolarized nor increased
[Ca2+]i in glomus cells
(Buckler 1997
; Cheng and Donnelly 1995
;
Lahiri et al. 1998
) (also Fig. 4C and Table
1). In contrast, dofetilide significantly depolarized glomus cells and
increased [Ca2+]i.
However, whether dofetilide affects hypoxia-induced depolarization and
[Ca2+]i in isolated
glomus cells remains to be investigated. These results demonstrate that
block of the HERG-like current, like hypoxia, causes depolarization and
increases [Ca2+]i in
glomus cells.
The role of O2-sensitive K+
channels in the transduction process at the carotid body has also been
questioned because known blockers of these channels (i.e.,
4-aminopyridine, TEA, and charybdotoxin) had no affect on basal sensory
discharge of intact carotid bodies (Buckler 1997;
Cheng and Donnelly 1995
; Lahiri et al.
1998
). In contrast, dofetilide significantly augmented baseline
sensory activity in the isolated carotid body in the present study. The fact that we have identified a HERG-like K+
current in glomus cells that is blocked by dofetilide suggests that these effects are mediated by effects on glomus cells themselves. In support of this idea, another report showed that millimolar concentrations of Ba2+ augment sensory discharge
of the carotid body (Donnelly 1997
). This augmentation
could be due to block of the HERG-like K+
current, because we found that mM Ba2+ inhibits
this current (Fig. 3C). In addition, the increase in nerve
activity in response to hypoxia was qualitatively and quantitatively comparable with that seen in response to dofetilide. Hypoxia
(PO2 = 38 ± 6 mmHg) augmented the baseline
sensory discharge by 87 ± 6%. However, given the constraints of
the experimental conditions, we cannot rule out possible effects of
dofetilide on sensory nerve endings. Nonetheless, this is the first
study to show that block of a specific K+ channel
augments the sensory discharge of the intact carotid body. It should
also be noted that the concentration of dofetilide required to produce
augmentation of sensory discharge in the intact carotid body was
relatively high and the response did not reach a plateau even with 3 µM. This is not unexpected because isolated cells and whole organs
are very different preparations and the effective concentration near
the glomus cell could be quite different because of the hydrophobic
nature of dofetilide. It is possible that the effect of dofetilide did
not reach a plateau because it has nonspecific effects at higher doses.
Most importantly, the results from our physiological studies clearly
show that modulation of the HERG-like current has a significant effect
on the sensory discharge of the intact carotid body. This suggests that
this current could be involved in the initial depolarization that is linked to the expression of the hypoxic response.
From the results of the present study, we cannot say whether or not the
HERG-like current participates in chemosensing or is sensitive to
O2. However, recent studies suggest that the HERG channel protein is a prime candidate for O2
sensing. For example, the HERG protein contains a PAS domain that is
known to be sensitive to O2 in other proteins
(Pellequer et al. 1999). However, the mechanism by which
O2 modulates channel activity may not be
straightforward. One possibility is that hypoxia could directly
modulate K+ channel activity. Alternatively,
hypoxia could modulate K+ currents by affecting
the redox state of the cell and/or altering the level of reactive
oxygen species (ROS) (Acker 1994
). In line with this
idea, ROS have been shown to modulate the activity of HERG channels
expressed in Xenopus oocytes (Taglialatela et al. 1997
). Therefore it is possible that O2
chemosensing could involve effects of hypoxia on HERG either directly
or indirectly through changes in ROS. These effects on HERG channel
activity could contribute to the depolarization responsible for the
initiation of sensory activity. We are currently investigating these possibilities.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-25830 to N. R. Prabhakar and an American Heart Association Grant-in-Aid to E. Ficker. J. L. Overholt is a Parker B. Francis Fellow in Pulmonary Research.
Permanent address of H. Shams: Institut fur Physiologie, Ruhr-Universitat Bochum, 44780 Bochum, Germany.
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FOOTNOTES |
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Address for reprint requests: N. R. Prabhakar, Dept. of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4970.
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.
Received 15 July 1999; accepted in final form 4 November 1999.
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REFERENCES |
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