§
§
§
From the * Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612; Playfair Neuroscience Unit, Toronto Hospital Research Institute, Toronto, Ontario M5T 2S8, Canada; § Department of Physiology,
University of Toronto, Toronto, Ontario M5S 1A1, Canada; and
Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S
2S2, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product
(HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at
hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage
relation was half maximal at 59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test
pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive
(
14 vs.
39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+
blocked with Ki = ~40 µM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the
presence of HERG-like K+ channels for the ontogeny of microglia are discussed.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microglia are macrophage-like cells of the brain that
are capable of serving typical phagocytic functions.
However, excessive microglial activity may play a pernicious role in Alzheimer's disease, AIDS-associated dementia, and other diseases (Streit and Kincaid-Colton,
1995). There is a long-standing controversy regarding
the origin of microglia (reviewed by Theele and Streit, 1993
). Although it is generally accepted that they are
derived from mesoderm (del Rio-Hortega, 1932
; for a
contrary view, see Schelper and Adrian, 1986
; Fedoroff,
1995
), it is not clear whether they entered the fetal
brain directly from a distinct pool of myelomonocyte
stem cells or first entered the bloodstream as circulating monocytes (reviewed in Ling and Wong, 1993
). Microglia resemble macrophages both in the types of ion
channels they express and in their plasticity; i.e., the
ability to alter their pattern of ion channel expression
in response to their environment (reviewed in DeCoursey and Grinstein, 1998
). Under many conditions, macrophages express inward rectifier K+ channels. In rat
microglia in culture, we observed inward K+ currents,
but with properties quite distinct from inward rectifier, and closely resembling those of the human ether-à-go-go-related gene (HERG)1 product. To our knowledge, this
is the first report of HERG channels in any immune
cell, including monocytes, macrophages, and related cell lines. The presence of this novel channel type is
consistent with microglial ontogeny distinct from that
of bone marrow-derived circulating monocytes/macrophages.
HERG K+ channels have been the focus of intense interest after the discovery that HERG mutations contribute to the genetic heart disease "long QT syndrome"
(Curran et al., 1995). HERG (Warmke and Ganetzky,
1994
) has been identified as encoding IKr, a K+ channel
of human cardiac myocytes (Sanguinetti et al., 1995
). IKr has been characterized in cardiac myocytes (Shibasaki, 1987
; Sanguinetti and Jurkiewicz, 1990a
), but
mRNA for erg is present in a number of different tissues
(Wymore et al., 1997
). K+ currents closely resembling
HERG have been described in mammalian neuroblastoma cells (Arcangeli et al., 1995
; Faravelli et al., 1996
;
Hu and Shi, 1997
), quail neural crest cells (Arcangeli et al., 1997
), Xenopus oocytes (Bauer et al., 1996
), GH3
cells (Weinsberg et al., 1997
), and in the present study
in rat microglia. In this paper, we characterize the electrophysiological, kinetic, and pharmacological properties of the HERG-like K+ current in microglia. Comparison of the properties of this current with HERG reveals
general similarities, but also some apparent differences. We speculate that the HERG-like K+ channels in
microglia are closely related but not identical to HERG
channels. In a companion paper (Pennefather et al.,
1998
), we propose a simple kinetic model that describes
the gating of these channels.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microglia Cell Culture
Microglia were isolated from brain explants of 2-3-d-old Wistar
rats using a modified version of established protocols (see Schlichter et al., 1996, for detailed methods and references). In
brief, neopallial tissue was digested in minimal essential medium
containing 0.25% trypsin and 25 µg/ml DNAse I (all from Sigma
Chemical Co., St. Louis, MO), triturated, and centrifuged to remove cell debris. The pelleted cells were resuspended in complete culture medium (MEM, 5% horse serum, 5% fetal bovine serum, 50 µg/ml gentamicin), seeded into tissue culture flasks and fed on day 7. After 12 days, flasks were shaken (180 rpm, 15 h),
floating cells were replated, allowed to adhere 1.5-2 h, and then
gently shaken by hand for 5 min to remove any remaining astrocytes. At this stage, the cultures were >95% microglia, as determined by labeling all cells with nuclear dyes, acridine orange or
propidium iodide (Molecular Probes, Inc., Eugene, OR), the living or fixed microglia with isolectin B4 (Streit, 1990
), and the
fixed and permeabilized astrocytes with an antibody directed against glial fibrillary acidic protein (both from Sigma Chemical Co.). Thereafter, the weekly feedings were supplemented with supernatant collected from the mouse fibroblast cell line, LM 10-5 (gift of Dr. S. Fedoroff, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada), which secretes large amounts of CSF-1, a
well-known stimulus of microglia proliferation (Fedoroff et al.,
1993
).
After several weeks in culture, it was often possible to withdraw
the CSF-1 containing supernatant and continue to grow the cells
in complete culture medium for many passages. Inasmuch as the
cells continued to proliferate without added growth factors, we
call this a cell line. All cells in the present study were from the
line that we have called MLS-9. (We have confirmed the presence
and fundamental properties of the HERG-like current in two
other similarly derived cell lines.) The cells stained positive with
several microglia markers: 100% with isolectin B4 (Streit, 1990),
100% with DiI-acetylated LDL and Lucifer Yellow (markers for
microglial endocytosis and pinocytosis; Booth and Thomas, 1991
;
Giulian, 1997
), 98% with OX-42 antibody, and 99% with ED-1 antibody (Booth and Thomas, 1991
). They did not label with antibodies against the astrocyte protein, glial fibrillary acidic protein (0%), or the fibroblast protein, fibronectin (0%), under conditions that clearly stained astrocytes and fibroblasts in primary
mixed cultures. A manuscript further describing these properties
of the MLS-9 cells is in preparation.
Electrophysiology
For patch-clamp recording, adherent microglia cells were released by incubating for 15 min in citrate solution (130 mM
NaCl, 15 mM Na citrate, 10 mM HEPES, 10 mM d-glucose, pH
7.4), and then plated onto glass coverslips at least 2 h before recording. A coverslip bearing microglia was placed in a superfusion bath on the stage of an inverted microscope. Experiments
were carried out in two separate labs using slightly different
equipment and solutions. Most of the experiments on current kinetics were performed at the Rush Presbyterian St. Luke's Medical Center, while most of the pharmacological characterization
was performed at the Toronto Hospital Research Institute and
the University of Toronto. In Chicago, micropipettes were pulled
in several stages using a Flaming Brown automatic pipette puller
(Sutter Instruments Co., San Rafael, CA) from EG-6 glass obtained from Garner Glass Co. (Claremont, CA). Pipettes were
coated with Sylgard 184 (Dow Corning Corp., Midland, MI) and
heat-polished to a tip resistance measured in bath saline of typically 2-5 M. Both pipette and the initial bath solutions were filtered with 0.22-µm pore diameter filters (Millipore Corp., Bedford, MA). The current signal from the patch clamp (Axopatch
1A; Axon Instruments Inc., Burlingame, CA) was digitized and
stored in computer files for off-line analysis using Indec Laboratory Data Acquisition and Display Systems (Indec Corp., Sunnyvale, CA) and pCLAMP 6.0.3 (Axon Instruments Inc.). In Toronto, a pipette puller (PP83; Narashige USA, Inc., Greenvale, NY)
was used to fabricate electrodes from thick-walled borosilicate
glass capillaries (WPI, Sarasota, FL) that were neither fire polished nor coated and typically had resistances of 5-10 M
, and a
series resistance of 15-30 M
after breakthrough. An Axopatch
200A amplifier (Axon Instruments Inc.) was used to record currents and both series resistance and capacitance compensation
were performed using the patch-clamp circuitry before data were
digitized. Currents were acquired and analyzed using pCLAMP
6.0 software. All experiments were done at room temperature
(20-23°C).
Solutions
Solutions are listed in Table I. Most salts and buffers were purchased from Sigma Chemical Co. Methanesulfonate (MeSO3
)
salts were prepared by titrating methanesulfonic acid (Aldrich Chemical Co., Milwaukee, WI) with the appropriate cation hydroxide to make a 1-M stock solution from which the solutions
were prepared. Except where stated otherwise, liquid junction
potentials were not corrected. When used, correction was based
on calculated junction potentials between the bath, pipette, and
ground agar bridge solutions (see Barry and Lynch, 1991
). Except where noted, bath and pipette K+ salines contained MeSO3
as the principal anion. At the Toronto Hospital Research Institute and the University of Toronto, the bath and pipette K+ salines differed somewhat from those used at the Rush Presbyterian St. Luke's Medical Center, mainly in the use of aspartate
as the
intracellular anion (Table I). No differences in the properties of
the HERG-like currents were noted. In the absence of internal
ATP, some current rundown was seen during ~30-min recordings. For pharmacological experiments, 2 mM ATP was added to
the pipette solution to prolong a stable baseline. In some cells, an
outwardly rectifying, time-invariant current was observed, which
resembled a swelling-sensitive anion current previously described
in primary rat microglia. In both primary cells and the cell line, it
was inhibited by flufenamic acid and ran down within minutes in
the absence of ATP in the pipette. When calculating HERG current amplitudes for pharmacological studies, this time-invariant current was subtracted, either as the current remaining after the
HERG channel closed at very negative potentials or after maximal block by E-4031. E-4031 is a class III antiarrhythmic methanesulfonanilide drug (Sanguinetti and Jurkiewicz, 1990a
). The E-4031
used here was manufactured by Merck Research Labs (White
House Station, NJ).
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inward K+ Currents
General description.
Voltage-gated inward K+ currents
were observed in nearly every microglial cell studied.
The general appearance of these currents is illustrated
in Figs. 1 and 2. The families of whole-cell currents in
Fig. 1 were obtained in isotonic K+ solutions. When the
holding potential (Vhold) was 80 mV (Fig. 1 A), 300-ms depolarizing pulses did not elicit detectable time-dependent currents. However, when hyperpolarizing
pulses were applied from Vhold = 0 mV (Fig. 1 B), large
inward currents were observed that increased to a peak,
and then decayed more slowly. A simple interpretation is that the current is activated by hyperpolarization and
subsequently inactivates. No currents are seen during
pulses from Vhold =
80 mV (Fig. 1 A), because at large
negative potentials all of the channels are inactivated,
defined as "a refractory or inactivated condition from
which [the channel] recovers at a relatively slow rate"
(Hodgkin and Huxley, 1952
). As is discussed in more
detail in the companion paper (Pennefather et al.,
1998
), various terminologies have been used to describe HERG and related currents. In describing our
results, we will define activation as the fast onset of current with hyperpolarization, and inactivation as the
slower closing to a state Cs that follows this opening.
The term deactivation will be used to describe the
rapid closing to a state Cr that occurs at depolarized potentials. The following general conceptual scheme
(Scheme I), in which hyperpolarization favors Cr
O
and O
Cs transitions and depolarization favors the
reverse, will be used to describe the data.
|
|
|
[K+]o dependence of the currents.
Fig. 2 compares the
behavior of the HERG-like conductance during identical pulse protocols in standard (low K+) saline (A) and
high K+ saline (B). In both, the holding potential was 0 mV, where most channels are in the rapidly gating
closed state Cr, enabling rapid activation on hyperpolarization. In Fig. 2, the current at the beginning of
pulses to large negative potentials appears to be somewhat greater than the leak current. In addition, the
small outward currents in Fig. 2 B decayed as the channels deactivated at more positive potentials. Both observations suggest that some channels were already open
at Vhold = 0 mV. The K+ currents in most cells were
small in standard (low K+) saline, consistent with the
strong dependence of the maximal conductance of
HERG on external K+ concentration (Sanguinetti et
al., 1995; Schönherr and Heinemann, 1996
; Wang et
al., 1997
). For this reason, we explored the properties of this conductance mainly in high K+ saline.
K+ selectivity.
The apparent reversal potential, Vrev,
was 78.0 ± 7.9 mV (mean ± SD) in standard saline
solution ([K+]o = 4.5 mM) in eight cells selected for
relatively small leak currents (<10 pA at
100 mV) and
1.9 ± 4.8 mV (corrected for liquid junction potentials, but without leak correction) in 12 cells in KCl saline. Thus, the channels underlying the voltage- and
time-dependent currents activated on hyperpolarization are K+ selective.
Gating Kinetics and Voltage Dependence
Voltage dependence of activation.
Fig. 3 A shows the average normalized peak current-voltage relationship measured in K+ saline during families of voltage-clamp
pulses from Vhold = 0 mV. There is distinct inward rectification, with large inward and small outward currents.
In 48 cells with a capacity of 18.4 ± 5.6 pF (mean ± SD), the mean current at 120 mV was
565 ± 250 pA
(mean ± SD, without leak subtraction). This corresponds with an average chord conductance of 2.6 pS/
µm2 (assuming a capacitance of 1 µF/cm2). Because
the instantaneous current-voltage relationship was linear (see Fig. 5 B, below), the voltage dependence of
channel opening can be estimated directly from the
peak K+ conductance (gK) during voltage pulses. The
average peak gK-voltage relationship is plotted in Fig. 3
B, along with the best-fitting Boltzmann curve. The
midpoint of the curve was
59 mV, and the slope factor was 18.6 mV.
|
|
Steady state availability of the K+ conductance.
The voltage
dependence of availability (the converse of inactivation) of the K+ conductance was assessed by applying
test pulses to 120 mV from various holding potentials
(Fig. 4, A and B). The inward test currents have characteristic rising and falling phases as channels first activate and then inactivate. At large negative Vhold, no channels were available for activation by the step to
120 mV, and no inward test current was elicited. (In
terms of our model, nearly all channels were in Cs [inactivated] states and few were in the Cr [resting] state.)
When Vhold was positive to 0 mV, the availability was
maximal. The availability, evaluated as the normalized peak inward test current (I/Imax), is plotted against the
prepulse potential in Fig. 4 C. This figure illustrates hysteresis in the availability measured when Vhold was made
progressively more positive (
) versus more negative
(
). Vhold was maintained for ~20 s at each potential
before the test pulse was applied. Evidently, 20 s is not
long enough for the system to achieve steady state, although the slowest time constants observed by direct
measurements of gating kinetics were only several seconds (see Fig. 8, below). Even when Vhold was maintained for 60 s at each potential, hysteresis was observed. Shifts in the voltage dependence of inactivation analogous to voltage shifts in various voltage-gated
channel properties seen in many cells after achieving
whole-cell configuration (Fenwick et al., 1982
; Fernandez et al., 1984
) cannot explain this phenomenon; similar hysteresis was observed during repeated measurements in the same cell. Therefore, the hysteresis must
reflect the existence of a previously unknown ultra-slow
gating process (Pennefather et al., 1998
).
|
|
|
|
Window currents.
The overlap we observed between
peak gK vs. voltage and availability vs. voltage relationships suggests the possibility of "window currents" that
might occur in intact microglia. It is evident in the
records in Fig. 4 A that a window current exists after
20 s at each Vhold just before applying a test pulse. That this current is due to HERG-like channels (as opposed
to leak or anion current) is evident from the reduction
of inward current upon the return to Vhold after each
test pulse, reflecting the inactivation of most K+ channels during the test pulse. The voltage dependence of
this window current is plotted in Fig. 4 D. Much smaller
window currents were seen when Vhold was made progressively more positive (Fig. 4 D, ). Like the availability curves in Fig. 4 C, the window current measurement
thus exhibited pronounced hysteresis. The peak on the
hyperpolarizing branch occurred at
40 mV and when converted to conductance was 14% of gK,max in that cell.
In four cells analyzed in this way, the window current at
40 mV averaged 13.6 ± 0.5% of gK,max (mean ± SD).
The model described in the next paper (Pennefather
et al., 1998
), using rate constants consistent with experimental observations, predicts a peak window conductance (at
36 mV) of ~12% of the maximal conductance.
The instantaneous current-voltage relation.
The instantaneous current-voltage relationship was determined
from experiments like the one in Fig. 5 A. The K+ conductance was activated by a brief pulse to 120 mV
from Vhold = 0 mV, and then the voltage was stepped to
a range of potentials. The test current at most potentials decayed rapidly as channels closed. The amplitude
of the "instantaneous" current was obtained by fitting
the decay with a single exponential and extrapolating to the start of the test pulse. When measured in K+ saline, the resulting instantaneous current-voltage relation was essentially linear between
80 and 80 mV (Fig.
5 B). Thus, the strong inward rectification of the macroscopic current is due mainly to voltage-dependent
gating and not to intrinsic rectification of the open
channel current.
Activation and deactivation kinetics.
When fitted by single exponentials, the time constants of channel opening and closing were moderately voltage dependent, as illustrated in Fig. 6. The activation time constant, act
(Fig. 6,
), measured during the turn-on of currents
during hyperpolarizing pulses (e.g., Figs. 1 and 2) was
~30 ms at
60 mV, decreasing e-fold in 54 mV to <10
ms at
120 mV.
|
The kinetics of inactivation (slow closing).
The time constants of inactivation (i) and recovery (
recovery) were
more strongly voltage dependent than those of activation (
act) and fast closing (
tail). Recovery from inactivation at moderate potentials was slow. Recovery was
evaluated in paired-pulse experiments like the one illustrated in Fig. 7. From Vhold = 0 mV, most of the
channels opened, and then were inactivated during a
pulse to
120 mV. The potential was returned to 0 mV
and after a variable interval a second pulse to
120 mV
was applied. The amplitude of the inward current during the second pulse reflects the recovery from inactivation that took place at 0 mV in the interval between
pulses. It is apparent that recovery required several seconds and was not yet complete after the largest interval illustrated (4 s). In other experiments, recovery was
measured at different potentials. The peak current during the second pulse as a function of the interval between pulses was fitted by a single exponential to obtain
recovery. At
20 to
40 mV, recovery seemed to be
slow and incomplete, and reliable data were not obtained. As shown in Fig. 8 (
),
recovery was strongly voltage dependent, decreasing on average e-fold in 35 mV.
Recovery from inactivation could be measured directly
in Cs+ saline as a rising outward current (Pennefather
et al., 1998
), which showed that
recovery decreased
steeply with depolarization also in Cs+ saline.
Pharmacological Sensitivity
Compared with IR, the HERG-like K+ currents in microglia were less sensitive to block by extracellular cations (Na+, Cs+, and Ba2+), and block was only weakly
time and voltage dependent. There was no obvious
time- or voltage-dependent block by Na+, and nearly
complete inactivation of inward K+ currents occurred
both in standard saline with 160 mM Na+ and in Na+-free K+ saline (Fig. 2, A and B). This is clearly distinct
from the effects of Na+ on IR channels. As seen in Fig.
9 A, when 10 mM Ba2+ was added to the bath the inward current was attenuated by >80% (n = 5), but the
decay time constant was only slightly faster than in the
control solution, in contrast to the more potent and rapid time-dependent block of IR currents in microglia
(Schlichter et al., 1996) and other cells. We cannot rule
out the possibility that some part of the effect of Ba2+
reflects the presence of a few IR channels in these cells.
La3+, which reportedly blocks HERG-like currents in a
voltage-dependent manner (Faravelli et al., 1996
), inhibited the microglial current (Fig. 9 B). There was little block at 3 µM, 37 ± 7% inhibition at 30 µM (mean ± SEM, n = 5), and 77 ± 5% inhibition at 100 µM (n = 8).
The methanesulfonanilide drug, E-4031, is a classic
blocker of HERG channels. HERG-like currents in microglia were sensitive to this drug, with substantial
block at 100 nM (Fig. 9 C). The Ki measured using the
illustrated protocol was estimated to be 37 ± 8 nM (n = 5).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Predominant K+ Current in Cultured Rat Microglia Was HERG-like and not Inward Rectifier
The presence of IR K+ currents in microglia from
mouse and rat has been demonstrated repeatedly
(Kettenmann et al., 1990; Banati et al., 1991
; Nörenberg et al., 1992
; Brockhaus et al., 1993
; Eder et al.,
1995a
; Ilschner et al., 1995
; Visentin et al., 1995
; Fischer et al., 1995
; Schlichter et al., 1996
), although
these studies also show that K+ channel expression
changes dramatically after treatment with various cytokines. IR channels in microglia exhibit classical properties of Kir2 channels, including high K+ selectivity,
[K+]o-dependent gating, voltage-dependent block by
Cs+, and voltage- and time-dependent block by Na+
and Ba2+ (Visentin et al., 1995
; Eder et al., 1995b
; Schlichter et al., 1996
). At a superficial level, the MLS-9 microglia preparation studied here appeared to have similar
K+ currents. However, closer inspection revealed several properties that clearly distinguish these currents
from traditional IR currents.
The inward K+ currents described here were affected
only by relatively high (millimolar) concentrations of
Ba2+ or Cs+. The time dependence of Ba2+ block was
distinctly weaker than for IR channels in microglia (Schlichter et al., 1996) and other cells. Cs+ was detectably permeant so that in isotonic Cs+ solution, distinct
inward and larger outward currents were observed (Pennefather et al., 1998
), as reported previously for
HERG (Schönherr and Heinemann, 1996
).
Macroscopic IR currents in many cells exhibit voltage- and time-dependent block by extracellular Na+
and slower, weak block by Ca2+ and Mg2+ (Biermans et
al., 1987), but little genuine inactivation. The decay of
IR currents in primary cultures of murine microglia
also appears to be due to Na+ block (Eder et al.,
1995a
). In contrast, the HERG-like currents described
here exhibited a strongly voltage-dependent inactivation (slow closing) process. Although
i was faster in
standard (high Na+) saline than in Na+-free K+ saline
(Fig. 8), the current decayed almost completely at large negative potentials in the complete absence of Na+.
Thus, the decay cannot be attributed to block by Na+.
A defining property of IR currents is their dependence on [K+]o. In a variety of cells, opening of IR
channels shows a nearly perfect dependence on [K+]o
such that the kinetics of opening and the conductance-voltage relation nearly superimpose when plotted as a function of V-EK (Almers, 1971; Hestrin, 1981
;
Harvey and Ten Eick, 1988; Silver and DeCoursey,
1990
; Pennefather et al., 1992
). Consistent with other
studies (Wang et al., 1997
; Yang et al., 1997
), activation of the HERG-like current in microglia appeared to be
somewhat faster in low than high [K+]o. In contrast,
lowering [K+]o slows the activation of IR channels at a
given voltage. Also in stark contrast to IR channels, we
found little effect of [K+]o on the position of the gK-V
relationship, consistent with previous studies of HERG-like currents in neuroblastoma (Arcangeli et al., 1995
),
and HERG in which V1/2 shifted only 30 mV when EK
was changed by 99 mV (Wang et al., 1997
).
Are HERG-like K+ channels expressed in microglia in situ? There are numerous reports of IR channels in microglia in primary culture. We did not observe IR currents in the MLS-9 cell line derived from microglia, and instead consistently observed HERG-like K+ currents. Clearly, the pattern of K+ channel expression is different in MLS-9 cells and in microglia in primary culture. This is not too surprising in light of the well-established propensity of cultured microglia to change their pattern of ion channel expression. The properties of the K+ currents reported previously in microglia identify them unambiguously as IR. An important question is whether HERG-like K+ channels are expressed in microglia in situ under any circumstances. In preliminary experiments (Cayabyab, F.S., and L.C. Schlichter, unpublished observations), we have explored the possible expression of HERG-like K+ current in cultured microglia soon after isolation, using E-4031 to distinguish it from classical IR. Large currents with properties very similar to those described herein were seen in a small number of microglia, under apparently specific conditions that have not yet been fully worked out. It may well be that this current was not discovered earlier in microglia because in most studies the conditions used would minimize the HERG-like current: low extracellular K+ (Fig. 2 A), and a negative Vhold that would inactivate HERG-like channels almost entirely (Fig. 1 A).
Implications for the ontogeny and functions of microglia.
There is a long-standing debate over whether microglia
originated in the brain from embryonic precursor cells,
or whether they are derived from circulating macrophages that migrated to the brain. A lack of delayed-rectifier K+ current in microglia, both in culture
(Kettenmann et al., 1990) and in brain slices (Brockhaus et al., 1993
), initially appeared to distinguish unstimulated microglia from macrophages. However, subsequent studies have shown that microglia can express
a delayed-rectifier current that is apparently identical
to that in macrophages. Both cell types have Kv1.3
mRNA transcripts, suggesting that this channel underlies the observed currents (Nörenberg et al., 1993
; DeCoursey et al., 1996
). Delayed rectifier currents appear
spontaneously in a fraction of cells (Korotzer and Cotman, 1992
; Schlichter et al., 1996
), and more consistently after exposure to astrocytes (Korotzer and Cotman, 1992
; Sievers et al., 1994
), to astrocyte-conditioned medium (Eder et al., 1997
), or to TeflonTM (Nörenberg
et al., 1993
), and after stimulation with PMA (Yoo et al.,
1996
), PMA and
-interferon (Visentin et al., 1995
), lipopolysaccharide (Nörenberg et al., 1994
; Illes et al.,
1996
), or granulocyte-macrophage colony stimulating
factor (Eder et al., 1995b
). Conversely, there are many
situations in which macrophages do not express delayed rectifier (Ypey and Clapham, 1984
; Gallin and
Sheehy, 1985
; Gallin and McKinney, 1988
; Nelson et
al., 1990
; DeCoursey et al., 1996
) and in which they express inward-rectifier channels (Gallin and Sheehy,
1985
; Randriamampita and Trautmann, 1987
; Gallin
and McKinney, 1988
; McKinney and Gallin, 1988
, 1990
;
DeCoursey et al., 1996
) like those described in microglia.
Possible roles for HERG in microglia.
Based on their near
ubiquity in microglia, IR channels are widely believed
to contribute most of the K+ conductance at the resting
membrane potential (Banati et al., 1991; Kettenmann
et al., 1990
; Korotzer and Cotman, 1992
; Nörenberg et
al., 1992
, 1994
; Fischer et al., 1995
; Schlichter et al., 1996
). The two stable membrane potentials that have been reported for microglia (
70 and
35 mV; Nörenberg et
al., 1994
) are near the K+ and Cl
equilibrium potentials (
85 and ~
30 mV). In cardiac muscle, HERG
currents contribute to repolarization during the long
action potential. There is no evidence that microglia
are excitable cells. However, at least under certain conditions, microglia possess a variety of channels that
upon activation would depolarize the membrane; e.g.,
purinergic receptor-gated (Kettenmann et al., 1993
; Illes et al., 1996
), Ca2+ (Colton et al., 1994
), Na+ (Korotzer and Cotman, 1992
), and anion (Schlichter et al.,
1996
) channels. The MLS-9 microglial cell line studied
here displayed little if any IR current and no delayed
rectifier K+ current. Inasmuch as the HERG-like current may be the only significant K+ current in this cell
line, and conceivably also in specific functional states of
microglial cells, it is significant that its voltage dependence of activation and inactivation predicts a standing window current at membrane potentials within the
physiological range (
50 to +20 mV). The substantial
window currents exhibited by HERG-like channels in
microglia would oppose depolarizing stimuli, such as
produced by purinergic stimulation.
Properties of HERG-like Currents in Microglia Compared with Other Cells
Pharmacological sensitivity.
La3+ blocks native HERG currents in cardiac muscle, IKr, (at 10-100 µM; Sanguinetti
and Jurkiewicz, 1990b), HERG-like currents in neuroblastoma cells (at 30 µM; Faravelli et al., 1996
), and HERG expressed in Xenopus oocytes (>90% inhibition
at 10 µM; Sanguinetti et al., 1995
). At least part of this
inhibition has been attributed to a shift in surface potential and, therefore, in the apparent voltage dependence of the current (Sanguinetti and Jurkiewicz, 1990b
; Sanguinetti et al., 1995
). The HERG-like current
in rat microglia was inhibited by La3+ with a Ki of ~40
µM. When expressed heterologously, the HERG channel (Trudeau et al., 1995
; Snyders and Chaudhary,
1996
), like cardiac IKr current (Sanguinetti and Jurkiewicz, 1990a
), is inhibited by the methanesulfonanilide drugs, E-4031 and dofetilide. Ki values reported for
E-4031 range from 10 nM in GH3 cells (Weinsberg et al., 1997
) and ferret atrial myocytes (Liu et al., 1996
) to
397 nM in guinea pig ventricular myocytes (Sanguinetti
and Jurkiewicz, 1990a
) and 588 nM for HERG expressed heterologously in Xenopus oocytes (Trudeau
et al., 1995
). Block by methanesulfonanilides exhibits
state dependence, interpreted as open-channel block
(Carmeliet, 1992
; Snyders and Chaudhary, 1996
; Spector et al., 1996b
), which most likely explains differences
in reported potency. In microglia, E-4031 blocked with
Ki = 37 nM, well within the range reported for HERG.
Inactivation is more steeply voltage dependent than activation.
The i-V,
recovery-V, and steady state availability
relationships of the HERG-like currents described here
all were steeply voltage dependent. A similarly steep
voltage dependence has been reported in all preparations in which HERG and related currents have been
studied (Table II). However, the midpoint (V1/2) is rather variable. The persistent hysteresis that we observed when measuring quasi-steady state availability
curves (Fig. 4 C) shows that quite different results can
be obtained if the measurement is made with different
voltage protocols. Depending on the history of the
measurement, V1/2 was
39 or
14 mV when measured in the same cells using 20-s prepulses. With 2-s
prepulses, V1/2 averaged 13.5 mV. Thus, the coupling of
the two closing pathways through the open state and/
or the existence of additional slowly equilibrating states
can dramatically influence this parameter, with V1/2
varying by 55 mV, depending on how it is measured.
Gating kinetics compared.
Both fast and slow gating
processes in microglia appear to be similar kinetically
to their counterparts in neuroblastoma cells (Arcangeli et al., 1995). Both gating processes are slower
than those measured in low K+ solutions for HERG
expressed in Xenopus oocytes (Trudeau et al., 1995
;
Schönherr and Heinemann, 1996
; Spector et al.,
1996a
; Wang et al., 1996
) or in HEK cells (Snyders and
Chaudhary, 1996
). However, the possible [K+]o dependence of gating complicates this comparison. The component of cardiac myocyte current believed to reflect
native HERG channels (IKr) appears to inactivate and
recover at least an order of magnitude faster in studies
at body temperature, 35-37°C (Shibasaki, 1987
; Sanguinetti and Jurkiewicz, 1990a
). The closest comparison is with HERG in Xenopus oocytes at 21-23°C with
[K+]o = 98 mM (Wang et al., 1997
), where
act,
tail, and
i appear similar to values reported here, but
recovery appears two to three times faster. Considering the differences in recording conditions, the currents described
here are kinetically similar to HERG and HERG-like
currents in other cells. The main difference seems to be
that HERG-like currents in microglia exhibit very slow
gating around
40 mV, which can lead to use-dependent phenomena.
![]() |
FOOTNOTES |
---|
Address correspondence to Tom DeCoursey, Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Fax: 312-942-8711; E-mail: tdecours{at}rpslmc.edu
Received for publication 5 August 1997 and accepted in revised form 18 March 1998.
Portions of this work were previously published in abstract form (Zhou, W., F.S. Cayabyab, P.S. Pennefather, L.C. Schlichter and T.E. DeCoursey. 1998. Biophys. J. 74:A109).The authors thank Claudia Eder for critically reading the manuscript, E. Wanke for generously providing a preprint, and P. Backx (Toronto Hospital Research Institute) for the E-4031.
This work was supported in part by Research Grant HL-52671 (T.E. DeCoursey) and Training Grant T32-HL07692 (W. Zhou), both from the National Institutes of Health, a Grant-in-Aid (NA-3182) from the Heart and Stroke Foundation of Ontario (L.C. Schlichter and P.S. Pennefather), and a grant from the Medical Research Council of Canada (L.C. Schlichter).
![]() |
Abbreviations used in this paper: E |
---|
K, Nernst potential for K+; gK, K+ conductance; HERG, human ether-à-go-go-related gene (erg) and its product; IR, inwardly rectifying K+ channel.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Almers, W. 1971. The Potassium Permeability of Frog Muscle Membrane. Ph.D. dissertation. University of Rochester, Rochester, NY. |
2. | Arcangeli, A., L. Bianchi, A. Becchetti, L. Faravelli, M. Coronnello, E. Mini, M. Olivotto, and E. Wanke. 1995. A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting potential of mammalian neuroblastoma cells. J. Physiol. (Camb.). 489: 455-471 [Abstract]. |
3. | Arcangeli, A., B. Rosati, A. Cherubini, O. Crociani, L. Fontana, C. Ziller, E. Wanke, and M. Olivotto. 1997. HERG- and IRK-like inward rectifier currents are sequentially expressed during neuronal development of neural crest cells and their derivatives. Eur. J. Neurosci. 9: 2596-2604 [Medline]. |
4. | Banati, R.B., D. Hoppe, K. Gottmann, G.W. Kreutzberg, and H. Kettenmann. 1991. A subpopulation of bone marrow-derived macrophage-like cells shares a unique ion channel pattern with microglia. J. Neurosci. Res 30: 593-600 [Medline]. |
5. | Barry, P.H., and J.W. Lynch. 1991. Liquid junction potentials and small cell effects in patch-clamp analysis. J. Membr. Biol. 121: 101-117 [Medline]. |
6. | Bauer, C.K., T. Falk, and J.R. Schwarz. 1996. An endogenous inactivating inward-rectifying potassium current in oocytes of Xenopus laevis. Pflügers Arch 432: 812-820 [Medline]. |
7. | Biermans, G., J. Vereecke, and E. Carmeliet. 1987. The mechanism of the inactivation of the inward-rectifying K current during hyperpolarizing steps in guinea-pig ventricular myocytes. Pflügers Arch 410: 604-613 [Medline]. |
8. | Booth, P.L., and W.E. Thomas. 1991. Evidence for motility and pinocytosis in ramified microglia in tissue culture. Brain Res. 548: 163-171 [Medline]. |
9. | Brockhaus, J., S. Ilschner, R.B. Banati, and H. Kettenmann. 1993. Membrane properties of ameboid microglial cells in the corpus callosum slice from early postnatal mice. J. Neurosci 13: 4412-4421 [Abstract]. |
10. | Carmeliet, E.. 1992. Voltage- and time-dependent block of the delayed K+ current in cardiac myocytes by dofetilide. J. Pharmacol. Exp. Ther. 262: 809-817 [Abstract]. |
11. |
Colton, C.A.,
M. Jia,
M.X. Li, and
D.L. Gilbert.
1994.
K+ modulation of microglial superoxide production: involvement of voltage-gated Ca2+ channels.
Am. J. Physiol
266:
C1650-C1655
|
12. | Curran, M.E., I. Splawski, K.W. Timothy, G.M. Vincent, E.D. Green, and M.T. Keating. 1995. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80: 795-803 [Medline]. |
13. | DeCoursey, T.E., and S. Grinstein. 1998. Ion channels and carriers in leukocytes. In Inflammation: Basic Principles and Clinical Correlates. 3rd ed. J.I. Gallin, R. Snyderman, D.T. Fearon, B.F. Haynes, and C. Nathan, editors. Raven Press, New York. In press. |
14. | DeCoursey, T.E., S.Y. Kim, M.R. Silver, and F.N. Quandt. 1996. III. Ion channel expression in PMA-differentiated human THP-1 macrophages. J. Membr. Biol. 152: 141-157 [Medline]. |
15. | del Rio-Hortega, P. 1932. Microglia. In Cytology and Cellular Pathology of the Nervous System. W. Penfield, editor. P.B. Hoeber, New York. 481-534. |
16. | Eder, C., H.-G. Fischer, U. Hadding, and U. Heinemann. 1995a. Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor. Pflügers Arch. 430: 526-533 [Medline]. |
17. | Eder, C., H.-G. Fischer, U. Hadding, and U. Heinemann. 1995b. Properties of voltage-gated potassium currents of microglia differentiated with granulocyte/macrophage colony-stimulating factor. J. Membr. Biol. 147: 137-147 [Medline]. |
18. | Eder, C., R. Klee, and U. Heinemann. 1997. Distinct soluble astrocytic factors induce expression of outward K+ currents and ramification of brain macrophages. Neurosci. Lett. 226: 147-150 [Medline]. |
19. | Faravelli, L., A. Arcangeli, M. Olivotto, and E. Wanke. 1996. A HERG-like K+ channel in rat F-11 DRG cell line: pharmacological identification and biophysical characterization. J. Physiol. (Camb.) 496: 13-23 [Abstract]. |
20. | Fedoroff, S. 1995. Development of microglia. In Neuroglia. H. Kettenmann, and B.R. Ransom, editors. Oxford University Press, New York. 162-181. |
21. | Fedoroff, S., C. Hao, I. Ahmed, and L.J. Guilbert. 1993. Paracrine and autocrine signalling in regulation of microglial survival. In Biology and Pathology of Astrocyte-Neuron Interactions. S. Fedoroff, B.H.J. Juurlink, and D. Doucette, editors. Plenum Publishing Corp., New York. 247-261. |
22. | Fenwick, E.M., A. Marty, and E. Neher. 1982. A patch clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J. Physiol. (Camb.) 331: 577-597 [Abstract]. |
23. | Fernandez, J.M., A.P. Fox, and S. Krasne. 1984. Membrane patches and whole-cell membranes: a comparison of electrical properties in rat clonal pituitary (GH3) cells. J. Physiol. (Camb.). 356: 565-585 [Abstract]. |
24. | Fischer, H.-G., C. Eder, U. Hadding, and U. Heinemann. 1995. Cytokine-dependent K+ channel profile of microglia at immunologically defined functional states. Neuroscience 64: 183-191 [Medline]. |
25. | Gallin, E.K., and L.C. McKinney. 1988. Patch-clamp studies in human macrophages: single-channel and whole-cell characterization of two K+ conductances. J. Membr. Biol. 103: 55-66 [Medline]. |
26. | Gallin, E.K., and P.A. Sheehy. 1985. Differential expression of inward and outward potassium currents in the macrophage-like cell line J774.1. J. Physiol. (Camb.) 369: 475-499 [Abstract]. |
27. | Giulian, D. 1997. Reactive microglia and ischemic injury. In Primer on Cerebrovascular Disease. K.M.A. Welch, L.R. Caplan, D.J. Reis, B.K. Siesjo, and B. Weir, editors. Academic Press, New York. 117-124. |
28. | Harvey, R.D., R.E. Ten, and Eick. 1988. Characterization of the inward-rectifying potassium current in cat ventricular myocytes. J. Gen. Physiol. 91: 593-615 [Abstract]. |
29. | Hestrin, S.. 1981. The interaction of potassium with the activation of anomalous rectification in frog muscle membrane. J. Physiol. (Camb.) 317: 497-508 [Abstract]. |
30. | Hodgkin, A.L., and A.F. Huxley. 1952. The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J. Physiol. (Camb.). 116: 497-506 [Medline]. |
31. | Hu, Q., and Y.L. Shi. 1997. Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma×glioma cells. Pflügers Arch 433: 617-625 [Medline]. |
32. | Illes, P., W. Nörenberg, and P.J. Gebicke-Haerter. 1996. Molecular mechanisms of microglial activation. B. Voltage- and purinoceptor-operated channels in microglia. Neurochem. Int. 29: 13-24 [Medline]. |
33. | Ilschner, S., C. Ohlemeyer, G. Gimpl, and H. Kettenmann. 1995. Modulation of potassium currents in cultured murine microglial cells by receptor activation and intracellular pathways. Neuroscience 66: 983-1000 [Medline]. |
34. | Kettenmann, H., R. Banati, and W. Walz. 1993. Electrophysiological behavior of microglia. Glia 7: 93-101 [Medline]. |
35. | Kettenmann, H., D. Hoppe, K. Gottmann, R. Banati, and G. Kreutzberg. 1990. Cultured microglial cells have a distinct pattern of membrane channels different from peritoneal macrophages. J. Neurosci. Res. 26: 278-287 [Medline]. |
36. | Korotzer, A.R., and C.W. Cotman. 1992. Voltage-gated currents expressed by rat microglia in culture. Glia. 6: 81-88 [Medline]. |
37. | Ling, E.-A., and W.-C. Wong. 1993. The origin and nature of ramified and amoeboid microglia: a historical review and current concepts. Glia. 7: 9-18 [Medline]. |
38. | Liu, S., R.L. Rasmusson, D.L. Campbell, S. Wang, and H.C. Strauss. 1996. Activation and inactivation kinetics of an E-4031-sensitive current from single ferret atrial myocytes. Biophys. J. 70: 2704-2715 [Abstract]. |
39. | McKinney, L.C., and E.K. Gallin. 1988. Inwardly rectifying whole-cell and single-channel K currents in the murine macrophage cell line J774.1. J. Membr. Biol. 103: 41-53 [Medline]. |
40. | McKinney, L.C., and E.K. Gallin. 1990. Effect of adherence, cell morphology, and lipopolysaccharide on potassium conductance and passive membrane properties of murine macrophage J774.1 cells. J. Membr. Biol. 116: 47-56 [Medline]. |
41. | Nelson, D.J., B. Jow, and F. Jow. 1990. Whole cell currents in macrophages: I. Human monocyte-derived macrophages. J. Membr. Biol. 117: 29-44 [Medline]. |
42. | Nörenberg, W., K. Appel, J. Bauer, P.J. Gebicke-Haerter, and P. Illes. 1993. Expression of an outwardly rectifying K+ channel in rat microglia cultivated on Teflon. Neurosci. Lett. 160: 69-72 [Medline]. |
43. | Nörenberg, W., P.J. Gebicke-Haerter, and P. Illes. 1992. Inflammatory stimuli induce a new K+ outward current in cultured rat microglia. Neurosci. Lett. 147: 171-174 [Medline]. |
44. | Nörenberg, W., P.J. Gebicke-Haerter, and P. Illes. 1994. Voltage- dependent potassium channels in activated rat microglia. J. Physiol. (Camb.). 475: 15-32 [Abstract]. |
45. |
Pennefather, P.S.,
W. Zhou, and
T.E. DeCoursey.
1998.
Idiosyncratic gating of HERG-like K+ channels in microglia.
J. Gen. Physiol
111:
795-805
|
46. | Pennefather, P., C. Oliva, and N. Mulrine. 1992. Origin of the potassium and voltage dependence of the cardiac inwardly rectifying K-current (IK1). Biophys. J. 61: 448-462 [Abstract]. |
47. | Randriamampita, C., and A. Trautmann. 1987. Ionic channels in murine macrophages. J. Cell Biol. 105: 761-769 [Abstract]. |
48. | Sanguinetti, M.C., C. Jiang, M.E. Curran, and M.T. Keating. 1995. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81: 299-307 [Medline]. |
49. | Sanguinetti, M.C., and N.K. Jurkiewicz. 1990a. Two components of cardiac delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents. J. Gen. Physiol 96: 195-215 [Abstract]. |
50. |
Sanguinetti, M.C., and
N.K. Jurkiewicz.
1990b.
Lanthanum blocks a
specific component of IK and screens membrane surface charge
in cardiac cells.
Am. J. Physiol.
259:
H1881-H1889
|
51. | Schelper, R.L., and E.K. Adrian. 1986. Monocytes become macrophages: they do not become microglia: a light and electron microscopic autoradiographic study using 125-iododeoxyuridine. J. Neuropathol. Exp. Neurol 45: 1-19 [Medline]. |
52. |
Schlichter, L.C.,
G. Sakellaropoulos,
B. Ballyk,
P.S. Pennefather, and
D.J. Phipps.
1996.
Properties of K+ and Cl![]() |
53. | Schönherr, R., and S.H. Heinemann. 1996. Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel. J. Physiol. (Camb.). 493: 635-642 [Abstract]. |
54. | Shibasaki, T.. 1987. Conductance and kinetics of delayed rectifier potassium channels in nodal cells of the rabbit heart. J. Physiol. (Camb.). 387: 227-250 [Abstract]. |
55. | Sievers, J., J. Schmidtmayer, and R. Parwaresch. 1994. Blood monocytes and spleen macrophages differentiate into microglia-like cells when cultured on astrocytes. Ann. Anat 176: 45-51 . |
56. | Silver, M.R., and T.E. DeCoursey. 1990. Intrinsic gating of inward rectifier in bovine pulmonary artery endothelial cells in the presence or absence of internal Mg2+. J. Gen. Physiol. 96: 109-133 [Abstract]. |
57. | Smith, P.L., T. Baukrowitz, and G. Yellen. 1996. The inward rectification mechanism of the HERG cardiac potassium channel. Nature. 379: 833-836 [Medline]. |
58. | Snyders, D.J., and A. Chaudhary. 1996. High affinity open channel block by dofetilide of HERG expressed in a human cell line. Mol. Pharmacol. 49: 949-955 [Abstract]. |
59. | Spector, P.S., M.E. Curran, A. Zou, M.T. Keating, and M.C. Sanguinetti. 1996a. Fast inactivation causes rectification of the IKr channel. J. Gen. Physiol 107: 611-619 [Abstract]. |
60. |
Spector, P.S.,
M.E. Curran,
M.T. Keating, and
M.C. Sanguinetti.
1996b.
Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Open-channel block by methanesulfonanilides.
Circ. Res
78:
499-503
|
61. | Streit, W.J.. 1990. An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4). J. Histochem. Cytochem. 38: 1683-1686 [Abstract]. |
62. | Streit, W.J., and C.A. Kincaid-Colton. 1995. The brain's immune system. Sci. Am 273: 54-61 [Medline]. |
63. | Theele, D.P., and W.J. Streit. 1993. A chronicle of microglial ontogeny. Glia 7: 5-8 [Medline]. |
64. | Trudeau, M.C., J.W. Warmke, B. Ganetzky, and G.A. Robertson. 1995. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269: 92-95 [Medline]. |
65. |
Visentin, S.,
C. Agresti,
M. Patrizio, and
G. Levi.
1995.
Ion channels
in rat microglia and their different sensitivity to lipopolysaccharide and interferon-![]() |
66. | Wang, S., M.J. Morales, S. Liu, H.C. Strauss, and R.L. Rasmusson. 1996. Time, voltage and ionic concentration dependence of h-erg expressed in Xenopus oocytes. FEBS Lett 389: 167-173 [Medline]. |
67. | Wang, S., S. Liu, M.J. Morales, H.C. Strauss, and R.L. Rasmusson. 1997. A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes. J. Physiol. (Camb.) 502: 45-60 [Abstract]. |
68. | Warmke, J.W., and B. Ganetzky. 1994. A family of potassium channel genes related to eag in Drosophila and mammals. Proc. Natl. Acad. Sci. USA 91: 3438-3442 [Abstract]. |
69. | Weinsberg, F., C.K. Bauer, and J.R. Schwarz. 1997. The class III antiarrhythmic agent E-4031 selectively blocks the inactivating inward-rectifying potassium current in rat anterior pituitary tumor cells (GH3/B6 cells). Pflügers Arch 434: 1-10 [Medline]. |
70. |
Wymore, R.S.,
G.A. Gintant,
R.T. Wymore,
J.E. Dixon,
D. McKinnon, and
I.S. Cohen.
1997.
Tissue and species distribution of
mRNA for the IKr-like K+ channel, erg.
Circ. Res
80:
261-268
|
71. |
Yang, T.,
D.J. Snyders, and
D.M. Roden.
1997.
Rapid inactivation
determines the rectification and [K+]o dependence of the rapid
component of the delayed rectifier K+ current in cardiac cells.
Circ. Res
80:
782-789
|
72. | Yang, T., M.S. Wathen, A. Felipe, M.M. Tamkun, D.J. Snyders, and D.M. Roden. 1994. K+ currents and K+ channel mRNA in cultured atrial cardiac myocytes (AT-1 cells). Circ. Res 75: 870-878 [Abstract]. |
73. | Yoo, A.S.J., J.G. McLarnon, R.L. Xu, Y.B. Lee, C. Krieger, and S.U. Kim. 1996. Effects of phorbol ester on intracellular Ca2+ and membrane currents in cultured human microglia. Neurosci. Lett 218: 37-40 [Medline]. |
74. | Ypey, D.L., and D.E. Clapham. 1984. Development of a delayed outward-rectifying K conductance in cultured mouse peritoneal macrophages. Proc. Natl. Acad. Sci. USA. 81: 3083-3087 [Abstract]. |