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J Biol Chem, Vol. 274, Issue 26, 18387-18392, June 25, 1999
From the Departments of Recent work has indicated that sigma receptor
ligands can modulate potassium channels. However, the only sigma
receptor characterized at the molecular level has a novel structure
unlike any other receptor known to modulate ion channels. This 26-kDa
protein has a hydropathy profile suggestive of a single
membrane-spanning domain, with no apparent regions capable of G-protein
activation or protein phosphorylation. In the present study patch clamp
techniques and photoaffinity labeling were used in DMS-114 cells (a
tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligands N-allylnormetazocine (SKF10047), ditolylguanidine, and
(±)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current
(IK). Iodoazidococaine (IAC), a high affinity
sigma receptor photoprobe, produced a similar inhibition in
IK, and when cell homogenates were illuminated
in the presence of IAC, a protein with a molecular mass of 26 kDa was
covalently labeled. Photolabeling of this protein by IAC was inhibited
by SKF10047 with half-maximal effect at 7 µM. SKF10047 also inhibited IK with a similar
EC50 (14 µM). Thus, physiological responses
to sigma receptor ligands are mediated by a protein with the same
molecular weight as the cloned sigma receptor. This indicates that ion
channel modulation is indeed mediated by this novel protein.
Physiological responses were the same when cells were perfused
internally with either guanosine 5'-O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism.
Sigma receptors are widely distributed in neuronal and nonneuronal
tissue and are distinguished by their ability to bind a broad range of
chemically unrelated ligands, including (+)-opiates, neuroleptic drugs,
ditolylguanidine (DTG),1 and
phencyclidine-related compounds (1-3). Although the biological activity of ligands suggests that sigma receptors may be involved in
behavioral, psychological, and motor functions (1-3), the cellular
actions of sigma receptors are poorly understood. Recent studies in
melanotrophs (4) and the neurohypophysis (5) showed that sigma receptor
ligands inhibit voltage-activated potassium current
(IK), but the signal transduction pathways
associated with sigma receptor activation remain unknown.
Molecular characterization of sigma receptors has raised intriguing
questions about how these receptors generate cellular responses. The
high affinity sigma receptor photoprobe iodoazidococaine (IAC) has
labeled a 26-kDa protein in rat liver, rat brain, and human placenta
(6). Cloning studies have confirmed that both human and rodent sigma
receptor cDNAs encode a 25.3-kDa protein (7-10). The protein
encoded for by these cDNAs binds sigma receptor ligands, but
physiological responses of expressed sigma receptors have yet to be demonstrated.
The amino acid sequence deduced from these clones is not easily
reconciled with a physiological function of ion channel modulation. The
vast majority of receptors that couple to separate ion channel proteins
contain seven putative membrane-spanning segments and require
G-proteins for signal transduction (11). Evidence both for (1, 4) and
against (12) a role for G-proteins in sigma receptor responses has been
presented, but hydropathy analysis of the deduced sigma receptor
sequence indicated that this protein contains a single putative
membrane-spanning domain, with no regions known to interact with
G-proteins. In fact, the proteins encoded for by sigma receptor
cDNAs are novel. There are no vertebrate homologues, and the only
known proteins with significant homology are fungal sterol isomerases.
Furthermore, the sigma receptor contains an endoplasmic reticulum
retention sequence. Thus, the structure of the sigma receptor raises
the question of whether it is found on the plasma membrane and whether
it is physically capable of modulating ion channels.
A number of clonal cell lines contain sigma receptors (13-16). One of
these, DMS-114, is derived from human small cell lung carcinoma and has
been shown to release neurohypophysial peptides (17, 18). Because our
own work on sigma receptors began in the neurohypophysis (5), we became
interested in using DMS-114 cells to investigate molecular aspects of
sigma receptor function in greater detail. We found that DMS-114 cells
are amenable to both photolabeling and patch clamp recording. This
enabled us to test the hypothesis that the 26-kDa protein is involved
in the modulation of IK by sigma receptor
ligands. Further experiments showed that G-proteins do not mediate this
response. Thus, this novel receptor protein employs a signal
transduction mechanism not yet encountered in the ligand-induced
modulation of ion channels.
Cell Culture--
DMS-114 cells were obtained from the ATCC,
Manassas, VA and maintained in Waymouth Medium 752/1 (ICN Biomedicals,
Costa Mesa, CA) with 10% bovine calf serum (Life Technologies, Inc.).
Flasks were incubated at 37 °C in 5% CO2, 95% air and
subcultured regularly by mechanical dissociation.
Photolabeling--
[125I]IAC was prepared
according to Kahoun and Ruoho (6). For photolabeling, DMS-114 cells
were pelleted by low speed centrifugation (200 × g)
and resuspended in phosphate-buffered saline (PBS) diluted 10-fold in
distilled water. (PBS had the following composition: 140 mM
NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.3.) Recombinant DNase (1 IU) was
then added, and cells were homogenized with a Teflon pestle. The
homogenate was resuspended in PBS and divided into 100-µl aliquots.
Sigma receptor ligands such as SKF10047 were then added, and the
mixture was incubated for 30 min on ice. [125I]IAC (1 nM) was added, and the incubation was continued for an additional 7.5 min, at which point illumination was then performed for
5 s with a high pressure AH-mercury lamp. Proteins were separated by SDS-polyacrylamide gel electrophoresis (12% acrylamide) and scanned
for [125I]IAC photolabeling on a PhosphorImager.
Permanent autoradiograms were developed on x-ray film for all
experiments included in this study.
Electrophysiology--
DMS-114 cells were plated on coverslips
for voltage clamp recording. 2 h prior to recording, coverslips
were transferred from the CO2/air incubator to a
superfusion chamber containing physiological salt solution (115 mM NaCl, 4.0 mM KCl, 1.25 mM
NaH2PO4, 26 mM NaHCO3,
2 mM CaCl2, 1 mM MgCl2,
and 10 mM glucose, pH 7.4) saturated with 95%
O2, 5% CO2. Individual cells were visualized
with an upright differential interference contrast microscope (Diastar, Leica Microsystems, Inc., Buffalo, NY) and a × 40 water
immersion, long working distance objective.
Voltage clamp recordings were made with an EPC-9 patch clamp amplifier
(InstruTECH Corp., Port Washington, NY) interfaced to a MacIntosh
computer. Whole-cell currents were recorded using patch pipettes filled
with 130 mM KCl, 10 mM EGTA, 2 mM
MgCl2, 4 mM MgATP, 100 µM NaGTP,
and 10 mM HEPES, pH 7.3. Pipettes were fabricated from thin
walled borosilicate glass, and the pipette shanks were coated with
Sylgard to reduce electrode capacitance (19). Prior to approaching the
cell membrane, pipette resistances typically ranged from 4 to 8 megaohms. Immediately after breaking in, cell capacitance and series
resistance were determined with the transient cancellation capability
of the EPC-9. In cases where the series resistance exceeded 15 megaohms, this value was partially compensated electronically.
Drug Application--
Sigma receptor ligands (other than IAC)
were obtained from Research Biochemicals, International (Natick, MA).
These compounds were dissolved in physiological buffer and added to the
bathing solution by direct pipette injection or through the use of a
simple gravity-feed system (rate, ~2 ml/min). Prior to the addition
of drugs, IK was recorded at 15-s intervals for
1-3 min to obtain a stable base line. IK was
also recorded after the removal of any drug(s) to demonstrate viability
of the cell and recovery of current to base line. In experiments
conducted using highly lipophilic drugs such as
(±)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin (PPHT), the agent was first dissolved in Me2SO and then
diluted into physiological saline to obtain the desired final drug
concentration. The concentration of Me2SO never exceeded
0.1%; this vehicle, without drug(s), was tested previously and found
to have no effect on IK (20).
Data Analysis--
Current recordings were analyzed on a
MacIntosh computer with the computer program Pulse + PulseFit
(InstruTECH Corp.). This program was used to fit current decays to
exponential functions. The computer program Origin (Microcal,
Northampton, MA) was used on a personal computer to fit
concentration-response data to the following function:
E = (100 Inhibition of Potassium Current by Sigma Receptor
Ligands--
DMS-114 cells displayed an outward current in response to
depolarizing test pulses under voltage clamp (Fig.
1). Voltage steps from
Whole-cell IK was also potently inhibited by the
sigma receptor ligand IAC (Fig. 1B). A radiolabeled version
of this agent was originally developed as a photoaffinity label for
neuronal sigma receptors (6, 14). Although significant inhibition of
IK was seen with 0.01 µM IAC, 1 µM IAC inhibited IK by more than
90%. The observation that IAC modulates IK
indicates that this compound is a potent sigma receptor agonist.
To address the issue of ligand selectivity, additional chemically
unrelated sigma receptor ligands were tested for inhibition of
IK. SKF10047 is the ligand for which sigma
receptors were initially named (22). Fig. 1 demonstrates that this
compound inhibits IK in a
concentration-dependent manner, and Fig.
2 shows that this effect is reversible.
Like SKF10047, DTG binds to sigma receptors with a
KD in the nanomolar range, and its affinity for members of other receptor families is on the order of 1000-fold lower
(1). Fig. 2 demonstrates that DTG reduced IK
with an efficacy and potency similar to SKF10047. Fig. 2 shows a
similar result with PPHT, a drug that binds both sigma receptors and
dopamine receptors (23) and that has recently been shown to modulate neurohypophysial IK (5, 20). Thus, four
compounds known to bind sigma receptors, IAC, SKF10047, DTG, and PPHT,
all inhibit IK in DMS-114 cells.
Photolabeling of Sigma Receptors--
[125I]IAC
covalently labels sigma receptors upon illumination and was used to
identify the receptor as a 26-kDa protein in rat brain and liver and in
human placenta (6). The labeling of this protein in each tissue could
be blocked by haloperidol as well as other sigma receptor ligands. As
noted above, IAC inhibited IK (Fig. 1). When
DMS-114 cells were homogenized and illuminated in the presence of
[125I]IAC, subsequent SDS-polyacrylamide gel
electrophoresis and phosphorimaging demonstrated labeling of a protein
with an apparent molecular mass of 26 kDa (Fig.
3). Photolabeling of the 26-kDa band in
DMS-114 cells was inhibited by two sigma receptor ligands, haloperidol (lane 2) and SKF10047 (lanes 4-6). This
indicates that DMS-114 cells contain a protein with a similar molecular
weight and that this protein has binding properties similar to
previously characterized sigma receptors.
A progressive increase in the block of [I125]IAC
photolabeling of the 26-kDa band was evident as the SKF10047
concentration increased from 1 to 100 µM (Fig.
4, top). The inhibitory effect
of SKF10047 on the photolabeling of this band was plotted
simultaneously with the inhibitory effect of SKF10047 on
IK (Fig. 4, bottom). These effects
had similar concentration dependences. When these data were fitted to a
single-site saturation equation (see "Experimental Procedures"), an
EC50 of 7 ± 3 µM was obtained for the
inhibition of photolabeling, and an EC50 of 14 ± 3 µM was obtained for the inhibition of
IK. These two values are statistically
indistinguishable, indicating that the inhibition of
IK is mediated by the 26-kDa sigma receptor
identified by photolabeling. This is an important result because it
links the 26-kDa sigma ligand binding protein to the functional
response of IK modulation.
Interestingly, the overall efficacy of SKF10047 appears to be identical
for the two end points of inhibition of photolabeling and inhibition of
IK. At 100 µM, SKF10047 reduced
whole-cell IK to 24 ± 5% of control
(n = 3); this same concentration of SKF10047 reduced
[125I]IAC photolabeling to 31 ± 8% of control
(n = 3). This suggests that full occupation of sigma
receptor binding sites produces a nearly complete block of
IK.
Transduction Mechanism--
Receptor-mediated modulation of
voltage-gated ion channels often reflects a shift in the voltage
dependence of the channel. Although this feature is not diagnostic of a
particular transduction mechanism, it is still widely regarded as
important. We therefore examined the voltage dependence of inhibition
of IK by PPHT by varying test pulses used to
activate IK from
DMS-114 cell IK exhibits a weak
voltage-dependent inactivation, as indicated by the decay
of whole-cell current (Fig. 1), and it was already noted above that the
time constants for activation and inactivation remained the same during
challenges with sigma receptor ligand. We also examined inactivation of
IK by varying the voltage at which cells were
held prior to depolarizing test pulses (to 10 mV). PPHT produced no
shifts in the voltage dependence of inactivation (data not shown).
These results indicate that sigma receptor-mediated modulation of
IK is not the result of shifts in voltage dependence.
Extensive literature on receptor-mediated modulation of ion channels
has shown that in most instances such responses are mediated by
G-proteins (11, 24, 25). However, the deduced amino acid sequence of
sigma receptors does not fit with a G-protein-coupled receptor motif
(7-10). There have been reports that the binding activity of sigma
receptors can be altered by GTP and guanosine nucleotides and that
sigma receptor-mediated responses are attenuated by cholera toxin and
GDP
Base-line currents were first collected for at least 3 min after break
in, allowing ample time for a molecule of this size to perfuse the cell
interior (26). G-protein-mediated responses in neurons have been shown
to be attenuated 75% by this mode of GDP These studies demonstrate that sigma receptor activation reduces
voltage-dependent IK by binding to a
protein with a molecular mass of 26 kDa. Four chemically distinct sigma
receptor ligands (SKF10047, DTG, PPHT, and IAC) inhibited
IK in a reversible,
concentration-dependent fashion. IAC also photolabeled a
protein with a molecular mass of 26 kDa, and this photolabeling was
blocked by concentrations of SKF10047 similar to those that inhibited
IK in patch clamp recordings. These studies
provide the first link of a protein with sigma ligand binding activity
to K+ channel modulation. Thus, despite the presence of a
putative endoplasmic reticulum sequence and the absence of putative
N-glycosylation sites (7-10), the physiological function of
the sigma receptor indicates a location in or near the plasma membrane.
The modulation of voltage-dependent K+ channels
by membrane-bound receptors has been extensively studied in many
systems. In general, transduction of these responses requires the
activation of a G-protein (11, 24, 25). Our observation that the
inhibition of IK is mediated by a receptor with
an apparent molecular mass of only 26 kDa is difficult to reconcile
with the hypothesis that G-proteins are involved in the transduction of
this response. Many G-protein-coupled receptors are similar in size to
rhodopsin, with molecular weights of roughly 40-50 kDa (plus
carbohydrate) (29). Although G-protein-coupled receptors with higher
molecular weights are quite common (30), homologous receptors with
molecular masses as low as 26 kDa have yet to be identified.
Furthermore, the seven transmembrane segments found in all known
G-protein-coupled receptors are inconsistent with the single
membrane-spanning segment implied by the hydropathy plots constructed
from the deduced sigma receptor amino acid sequence (7-10).
Efforts to establish a role for G-proteins in sigma receptor function
have met with mixed results. Cholera toxin reduced responses to sigma
receptor ligands in one study (4) but not in another (12). In
melanotrophs, the guanine nucleotide analogue GDP If G-proteins do not mediate the inhibition of
IK by sigma receptor activation, then these
results may indicate that sigma receptor-mediated signal transduction
depends on other molecular factors. An interesting possibility is that
sigma receptors alter K+ channel activity through a direct
protein-protein interaction with the channel, analogous to the actions
of auxiliary It is noteworthy that DMS-114 cells are tumor cells derived from a
neuroendocrine progenitor (34). These cells retain many properties of
an excitable cell line, including the expression of
voltage-dependent ion channels (35, 36). The coupling
between sigma receptors and IK described here in
DMS-114 cells is very similar to that found in neuroendocrine
neurohypophysial nerve terminals (5). Furthermore, DMS-114 cells
exhibit ectopic release of the neurohypophysial hormone vasopressin
(17, 18). The coexpression of these distinctive properties in both
DMS-114 cells and the neurohypophysis may reflect a link in the
mechanisms of regulation of different cellular functions that is
operating both in this tumor cell line and in the native hypothalamic
neurohypophysial system. In vivo, tumor-related secretion of
ectopic neurohypophysial hormones is responsible for inducing
derangements of fluid and sodium homeostasis in as many as 30% of
patients with primary small cell lung carcinoma (37). The finding that
sigma receptors are functionally linked to membrane excitability in
such cells indicates that this protein may represent a useful
therapeutic target for the curtailment of ectopic hormone secretion.
We thank Dr. Bradford Schwartz for providing
the DMS-114 cells and Drs. Gerard Ahern and Sujata Swaminathan for help
in the design of the experiments.
*
This work was supported in part by National Institutes of
Health Grant NS30016.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.
§
Trainee in the Clinical Investigator Pathway at the University of Wisconsin.
¶
Supported by National Institutes of Health Physician Scientist
Training Grant 5-T32CA09614-10.
2
P. J. Lupardus, R. A. Wilke, Y. Chen,
R. E. Ruoho, and M. B. Jackson, submitted for publication.
The abbreviations used are:
DTG, ditolylguanidine;
IK, voltage-activated
potassium current;
IAC, iodoazidococaine;
G-protein, guanine-nucleotide
binding protein;
PBS, phosphate-buffered saline;
PPHT, (±)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin;
GDP
Sigma Receptor Photolabeling and Sigma Receptor-mediated
Modulation of Potassium Channels in Tumor Cells*
§,¶,
,
Medicine, Physiology,
and ** Pharmacology, University of Wisconsin School of Medicine,
Madison, Wisconsin 53706
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Emax)/(1 + C/EC50) + Emax, where E denotes IK expressed as a percent
of predrug control, C denotes the concentration of ligand in
the superfusion medium, Emax denotes response at
saturating ligand, and EC50 denotes the concentration of
drug producing a half-maximal effect (defined by the condition (E 50)/Emax = 1/2).
Conductance-voltage plots were fitted to the following Boltzmann
function: G = Gmin + (Gmax Gmin) (1 + e(V
V1/2)/k)
(21). All four parameters, Gmin,
Gmax, k, and V1/2 were varied to achieve the best fit. Gmin and
Gmax represent conductance asymptotes at
negative and positive prepulse potentials, respectively, k
is the steepness factor, and V1/2 is the midpoint
for voltage dependence. Simple statistical analyses were performed
using the program Microsoft Excel. When arithmetic means were computed,
they were presented with the S.E. All null hypotheses were subjected to
a Student's t test, and a level of p < 0.05 was considered statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 to 10 mV
rapidly activated this current, which showed a slight exponential
decline as the potential was held constant for 500 ms. Tail currents
reversed direction at the K+ equilibrium potential, and
this value shifted appropriately with changes in external
K+ concentration, indicating that the outward current was
carried by K+ (n = 3, data not shown). When
DMS-114 cells were exposed to the sigma receptor agonist SKF10047,
whole-cell IK was inhibited in a
concentration-dependent fashion (Fig. 1A). Kinetic
analysis revealed that the current amplitude was uniformly reduced by
this agent and that the time constants for channel activation and
inactivation remained unaltered (control: 
act = 9.7 ± 0.6 ms, inact = 3.8 ± 1.7 s; 10 µM SKF10047: act = 9.7 ± 3.2 ms,
inact = 3.8 ± 1.0 s; n = 3).
View larger version (18K):
[in a new window]
Fig. 1.
Sigma receptor ligands inhibit
IK. Whole-cell
IK was measured in voltage-clamped DMS-114
cells. The membrane potential was held at 80 mV, stepped to 10 mV for
500 ms, and then returned to the holding potential. Outward current was
rapidly activated and then decayed slightly over time. A, a
concentration-dependent reduction in
IK was observed during exposure to the sigma
receptor ligand, SKF10047. B, IAC produced a similar
reduction in IK but was significantly more
potent.
View larger version (18K):
[in a new window]
Fig. 2.
Time course of sigma receptor-mediated
inhibition of IK. Current was
recorded at 15-s intervals before, during, and after challenge with
agonist. The voltage pulse paradigm was identical to that of Fig. 1.
Current was normalized to predrug control. Superfusion with all three
agents uniformly reduced IK. Each agonist was
studied in at least three cells. A representative tracing is shown for
each. All effects were reversed when agonist was washed from the
medium.
View larger version (52K):
[in a new window]
Fig. 3.
[125I]IAC photolabeling of
membrane proteins and inhibition of photolabeling by two chemically
unrelated sigma receptor ligands. A, Coomassie Blue
staining of an SDS-12% polyacrylamide gel of homogenate prepared from
DMS-114 cells. B, the homogenate had been photolabeled with
1 nM [125I]IAC, and an autoradiogram of the
gel revealed radiolabel incorporation. The labeling of a 26-kDa protein
was selectively inhibited by sigma receptor ligands. Treatments were as
follows: lane 1, control; lane 2, 10 µM haloperidol; lane 3, control; lane
4, 1 µM SKF10047; lane 5, 10 µM SKF10047; lane 6, 100 µM
SKF10047.
View larger version (29K):
[in a new window]
Fig. 4.
Concentration dependence of photolabeling
inhibition and IK inhibition. An
experimental protocol similar to that of Fig. 3 was used to examine the
concentration dependence of an SKF10047 block of
[125I]IAC photolabeling (top).
IK was also measured in the presence of various
SKF10047 concentrations ([SKF](µM)), using the pulse
protocol from Fig. 1. Left axis, the concentration
dependence of current reduction, plotted as normalized
IK averaged over three experiments (closed
squares); right axis, the concentration dependence of
photolabeling inhibition (closed circles). For both effects,
the best fitting curves are shown as dotted lines (see text
for parameter values).
60 to 30 mV. Current was
recorded before and after a 3-min exposure to 30 µM PPHT. At all voltages tested, PPHT reduced IK by
approximately proportional amounts, suggesting that sigma receptor
ligands do not produce their inhibitory effect on K+
channels by shifting the voltage dependence of activation. Current was
converted to conductance using the relation, GK = IK/[V 
K],
where GK is K+ conductance and
K is the Nernst potential for K+ computed
for the bathing and patch pipette solution compositions. The plots of
GK versus pulse potential are shown
in Fig. 5 along with best fitting
Boltzmann functions (see "Experimental Procedures"). Under control
conditions, the conductance-voltage plot was characterized by a
steepness factor (k) of 10 ± 1 mV and a voltage
midpoint (V1/2) of 6 ± 1 mV. After exposure to PPHT, neither the steepness factor (6 ± 4 mV) nor the voltage midpoint (9 ± 4 mV) had been altered (n = 3).
The modulation of IK in neurohypophysial nerve
terminals by sigma receptor ligands exhibits a similar voltage
independence (5, 20).
View larger version (13K):
[in a new window]
Fig. 5.
Potassium conductance versus
voltage. Cells were held at 80 mV and interrupted at 5-s
intervals with 500-ms pulses to each of the voltages shown. Current was
then converted to conductance (see text) and plotted as a function of
voltage (open circles). After a 3-min exposure to 30 µM PPHT (closed circles), the cells were again
subjected to a repeat series of voltage pulses. Each data point
represents the mean current from three experiments.
S (1, 4), but another study showed that cholera toxin had no
effect (12). Both the size (1) and deduced amino acid sequence (7-10)
of sigma receptors are difficult to reconcile with that of known
G-protein-coupled receptors. We therefore tested the role of G-proteins
by adding GDPS (100 µM) to the patch pipette filling
solution and allowing it to diffuse into the cell interior during
whole-cell recordings.
S addition 2 min after
break in (27). We found that responses to SKF10047 were not reduced by
GDPS. Both 10 and 100 µM SKF10047 showed equal
efficacy for the inhibition of IK regardless of
whether cells were perfused with 100 µM GDPS or 100 µM GTP (Fig. 6). It
therefore appears that sigma receptor-mediated modulation of IK can occur independently of G-protein
activation. Similar results have been obtained with sigma
receptor-mediated modulation of IK in rat
neurohypophysial nerve
terminals.2 In these
experiments GDPS also failed to block the modulation of
IK by SKF10047. The entry of the guanine
nucleotide GTP
S into neurohypophysial terminals was verified by
monitoring current through Ca2+-activated K+
channels. GTPS triggers the G-protein-mediated dephosphorylation of
this channel in the neurohypophysis (28), and 50 µM
GTPS added to the patch pipette filling solution reduced current to 79 ± 3% of the original level at break in, with a half-time of 29 ± 4 s (n = 11).
View larger version (29K):
[in a new window]
Fig. 6.
G-protein independence of sigma
receptor-mediated changes in IK.
IK was monitored before and after superfusion
with various concentrations of SKF10047, according to the same protocol
employed in Fig. 1. Control experiments were conducted with pipette
solution containing 100 µM GTP (open bars). A
parallel set of experiments was then conducted with GTP replaced by 100 µM GDP S in the patch pipette solution (closed
bars). At all concentrations studied, the effects of SKF10047 were
identical between GTP and GDPS. Data represent mean ± S.E. for
3-6 terminals.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S has been shown to
prevent the inhibition of IK by DTG (4). These
results may reflect the existence of another molecular species of sigma
receptor that is a member of the G-protein-coupled receptor family. In
the present study, cells perfused internally with GDPS exhibited the
same response to SKF10047 as control cells perfused internally with
GTP, and GDPS is known to be a very potent inhibitor of
G-protein-dependent processes (11, 24, 25, 27, 29). This
result therefore indicates that the recently cloned 26-kDa receptor
modulates K+ channels without coupling to G-proteins. In
this regard it is relevant that the concentration dependences of both
ligand binding and IK inhibition by SKF10047
were similar (Fig. 4). In contrast to G-protein-coupled receptors,
where spare receptors and other variations in the efficiency of
coupling can cause large discrepancies between the apparent
KD and the physiological EC50 (29), the
modulation of K+ channel function by sigma receptor ligands
appears to be tightly coupled to receptor binding.
subunits (31) and MinK proteins (32, 33), both of
which can modulate the function of voltage-gated K+
channels without forming channels themselves. Another possibility is
that sigma receptors interact with a protein kinase (by a
G-protein-independent mechanism) and that this enzyme modifies channel
function. These mechanisms have not been demonstrated previously in
ligand-induced ion channel modulation, indicating that sigma receptors
inhibit IK by a new transduction process.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Physiology, University of Wisconsin School of Medicine, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-9111; Fax: 608-265-5512; E-mail: mjackson{at}macc.wisc.edu.
ABBREVIATIONS
S, 5'-O-(2-thiodiphosphate);
GTPS, guanosine
5'-O-(3-thiotriphosphate).
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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 D. Fontanilla, M. Johannessen, A. R. Hajipour, N. V. Cozzi, M. B. Jackson, and A. E. Ruoho The Hallucinogen N,N-Dimethyltryptamine (DMT) Is an Endogenous Sigma-1 Receptor Regulator Science, February 13, 2009; 323(5916): 934 - 937. [Abstract] [Full Text] [PDF]
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 A. Pal, U. B. Chu, S. Ramachandran, D. Grawoig, L.-W. Guo, A. R. Hajipour, and A. E. Ruoho Juxtaposition of the Steroid Binding Domain-like I and II Regions Constitutes a Ligand Binding Site in the {sigma}-1 Receptor J. Biol. Chem., July 11, 2008; 283(28): 19646 - 19656. [Abstract] [Full Text] [PDF]
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 A. Pal, A. R. Hajipour, D. Fontanilla, S. Ramachandran, U. B. Chu, T. Mavlyutov, and A. E. Ruoho Identification of Regions of the {sigma}-1 Receptor Ligand Binding Site Using a Novel Photoprobe Mol. Pharmacol., October 1, 2007; 72(4): 921 - 933. [Abstract] [Full Text] [PDF]
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 E. Aydar, C. P. Palmer, and M. B. A. Djamgoz Sigma Receptors and Cancer: Possible Involvement of Ion Channels Cancer Res., August 1, 2004; 64(15): 5029 - 5035. [Abstract] [Full Text] [PDF]
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S. Zhang, S. Rajamani, Y. Chen, Q. Gong, Y. Rong, Z. Zhou, A. Ruoho, and C. T. January Cocaine Blocks HERG, but Not KvLQT1+minK, Potassium Channels Mol. Pharmacol., April 16, 2001; 59(5): 1069 - 1076. [Abstract] [Full Text]
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 P. J Lupardus, R. A Wilke, E. Aydar, C. P Palmer, Y. Chen, A. E Ruoho, and M. B Jackson Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP J. Physiol., August 1, 2000; 526(3): 527 - 539. [Abstract] [Full Text] [PDF]
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 M. Schaefer, U. F. Habenicht, M. Bräutigam, and T. Gudermann Steroidal Sigma Receptor Ligands Affect Signaling Pathways in Human Spermatozoa Biol Reprod, July 1, 2000; 63(1): 57 - 63. [Abstract] [Full Text]
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