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Vol. 289, Issue 3, 1447-1453, June 1999
Department of Pharmacology and Neuroscience Center, University of Colorado Health Sciences Center, Denver, Colorado
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The dye ruthenium red (RuR) has diverse experimental uses, including block of ion channels. RuR is a well described antagonist of one class of intracellular Ca2+ release channels, the ryanodine receptors, but recently this compound has also been identified as a putative blocker of voltage-gated calcium channels of the surface membrane involved in neurotransmitter release. Using electrophysiological methods, we have studied the action of RuR upon pure populations of neuronal voltage-gated ion channels heterologously expressed in Xenopus laevis oocytes. All four channel types studied, including class A (P/Q-type), class B (N-type), class C (L-type), and class E channels, are sensitive to RuR, with IC50 values ranging from 0.7 to 67.1 µM. Block of class C and class E channels most likely results from 1:1 binding of ruthenium red at a site in the extracellular entrance to the pore, resulting in obstruction of permeant ion flux through these channels. The mechanism of block of class A and class B channels is more complex, requiring binding of more than one molecule of RuR per channel.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Ruthenium
red (RuR) is a water-soluble hexavalent cation with several major uses
in biological research. The compound has been used as a stain in light
microscopic studies of polyvalent anionic materials such as plant
lectins, and RuR has been extensively used in combination with osmium
tetroxide as an electron-opaque dye for electron microscopic work. RuR
is also an inhibitor of a surprisingly wide range of
Ca2+-binding proteins (Charuk et al., 1990
).
These include the mitochondrial Ca2+ uniporter
(Moore, 1971), the Ca2+-ATPase of the
sarcoplasmic reticulum (Vale and Carvalho, 1973), troponin C (Charuk et
al., 1990), calsequestrin (Charuk et al., 1990), and calmodulin (Sasaki
et al., 1992).
RuR is also well known as an antagonist of ryanodine receptors. These
receptors are present in the intracellular membranes that bound
internal Ca2+ stores and are ion channels
responsible for signal-dependent release of stored
Ca2+ into the cytosol. RuR binds directly to
ryanodine receptors (Smith et al., 1988; Chen and MacLennan, 1994) and
blocks these channels by lodging in the pore (Ma, 1993). The apparent
affinity for RuR block of ryanodine receptors is in the range of 0.1 to
1 µM. Sensitivity of Ca2+ release to RuR is one
of the principal pharmacological means of distinguishing release via
ryanodine receptors from that by 1,4,5-bis inositol trisphosphate
receptors (Ehrlich et al., 1994).
Other kinds of ion channels are also affected by RuR.
Capsaicin-activated nonselective cation channels involved in
nociception are inhibited by submicromolar concentrations of RuR (Dray
et al., 1990; Bleakman et al., 1990). Inactivation of voltage-gated sodium channels is slowed by 1 to 10 nM RuR (Stimers and Byerly, 1982;
Neumcke et al., 1987). Various actions on potassium channels have been described for micromolar RuR, depending upon channel type and
whether RuR was applied extra- or intracellularly. Micromolar external
(Hirano et al., 1998) or internal (Wann and Richards, 1994) RuR blocks
large-conductance Ca2+-activated potassium
channels, internal RuR probably acting by antagonizing
Ca2+ binding. RuR has also been reported to
enhance the activity of other kinds of
Ca2+-activated potassium channels and of fast
potassium channels while not affecting delayed rectifier type potassium
channels (Lin and Lin-Shiau, 1996).
Like other Ca2+ binding proteins, including
ryanodine receptors, the voltage-gated Ca2+
channels of cell surface membranes have been reported to be sensitive to RuR (Tapia et al., 1985; Tapia and Velasco, 1997). Voltage-gated Ca2+ channels in neurons from snail (Stimers and
Byerly, 1982), rat (Hamilton and Lundy, 1995), and mouse (Lin and
Lin-Shiau, 1996), and in smooth muscle of guinea pigs (Hirano et al.,
1998) have all been reported to be blocked by RuR. In smooth muscle,
the principal voltage-gated Ca2+ channel is an
L-type channel sensitive to dihydropyridines, and this is presumably
the channel blocked by RuR. However, in neurons it has been reported
that N- and P/Q-type Ca2+ channels, but not
L-type Ca2+ channels, are antagonized by RuR
(Hamilton and Lundy, 1995).
These effects of RuR upon ion channels, particularly voltage-gated
Ca2+ channels, naturally have consequences for
synaptic transmission. For example, it has previously been shown that
RuR interferes with normal neurotransmission (Taipale et al., 1989) and
it is believed that RuR block of N- and P/Q-type Ca2+
channels is at least partly responsible for RuR inhibition of neurotransmission (Tapia et al., 1985; Hamilton and Lundy, 1995). RuR
has also been reported to block a form of
non-N-methyl-D-aspartate receptor-dependent synaptic plasticity (Wang et al., 1996b), an effect
that may be attributable to Ca2+ channel block,
ryanodine receptor block, or a direct action of RuR upon the
neurotransmitter release apparatus (Trudeau et al., 1996).
Clearly, the spectrum of RuR action is broad, which in some cases leads to uncertainty in attributing the effects of RuR to specific molecular targets. To clarify the role of RuR as a putative antagonist of neuronal voltage-gated Ca2+ channels, we have measured the sensitivity to RuR of pure populations of neuronal (classes A, B, C, and E) voltage-gated Ca2+ channels heterologously expressed in Xenopus laevis oocytes, and we have investigated the mechanism of the antagonism. Each of these four channel types was blocked by RuR, but with differing half-block (IC50) values. The mechanism of block is in general complex, but involves at least in part binding within the channel entrance and obstruction of current flow.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression of Ca2+ Channels in Xenopus
Oocytes.
The methods are modified from Methfessel et al. (1986)
and from Sather et al. (1993). Briefly, female Xenopus
laevis were anesthetized by ~30-min immersion in a 0.2%
tricaine methanesulfonate solution. The anesthetized frogs were placed
on a bed of ice for the surgical procedure, with a layer of damp paper
towels protecting the frogs' skin from ice burns. Ovarian tissue was
removed via an abdominal incision, the incision was sutured, and the
frogs returned to their housing for re-use in later experiments.
1A; Mori et al., 1991), pSPCBIII
(rabbit 1B; Fujita et al., 1993), pCARD3
(rabbit cardiac 1C;
1C-a; Mikami et al., 1989), pSPCBII-2 (rabbit
1E; Niidome et al., 1992), pSPCA1 (rabbit
skeletal muscle 2
;
2a; Mikami et al.,
1989), and pCaB2b (rabbit 
2b; Hullin et al.,
1992). cRNAs were injected in a 1:1:1 M ratio, with a total volume of
~50 nl of cRNA injected into each oocyte. Injected oocytes were
stored at 18°C for up to 4 weeks in ND96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2 1 MgCl2, 5 HEPES, pH
7.6 with NaOH) supplemented with 2.5 mM sodium pyruvate, 100 U/ml
penicillin, and 0.1 mg/ml streptomycin.
Two-Electrode Voltage Clamp Recording.
Whole cell
Ca2+ channel currents were recorded using a
standard two-electrode voltage clamp amplifier (model OC-725C; Warner Instruments, Hamden, CT). Glass microelectrodes typically had resistances of 0.3 M
and were filled with 3 M KCl. Membrane currents were filtered at 0.5 KHz and sampled at 1 KHz. Leak and capacitive currents were subtracted using a P/4 protocol. Nevertheless, capacitive currents were incompletely subtracted, owing to the intrinsic slowness
in clamping such large cells; the residual capacitive transients have
been deleted from the figures. Depolarizing voltage pulses of 150-ms
duration were delivered every 15 s during the experiments. The
extracellular solution was chloride-free and contained (in mM):
(nominally) 40 Ba(OH)2, 52 tetraethylammonium hydroxide, 5 HEPES, pH 7.4 using methane sulfonic acid. Addition of
methane sulfonic acid to adjust solution pH to 7.4 caused significant precipitation of Ba2+; this precipitate was
removed by filtration. Posthoc elemental analysis (Evergreen
Analytical, Wheat Ridge, CO) of the nominally 40 mM
Ba2+ solution revealed that the
Ba2+ concentration ranged from 8.7 to 11.7 mM; in
keeping with the extensive body of previous work utilizing this
solution, however, we have continued to refer to this solution as "40
mM Ba2+" throughout. A set of ten reservoirs,
containing control solution or control solution supplemented with
various concentrations of RuR, delivered a constant flow (~1 ml/min)
through the slot-shaped recording chamber (15-mm length × 3-mm
width × 3-mm depth).
Vm)/ba)]
1,
whereas that for inactivation was: G/Gmax = [1 + exp(Vm Vi)/bi]1,
where Vm is the membrane potential,
Va and
Vi are the midpoint voltages of the
activation and inactivation functions, and ba and
bi are the Boltzmann slope parameters for
activation and inactivation.
The fraction of the electric field traversed by RuR in reaching its
binding site was calculated using an equation of the following standard
form: fb = [1 + (KD/[RuR])
· exp(zVmF/RT)]1,
where fb is the fractional block,
KD is the apparent dissociation constant measured at 0 mV, [RuR] is the concentration of the blocker, z is the valence of RuR (+6), is the fractional electric distance from the outside of the membrane, F is the Faraday, R is the
gas constant, T is the absolute temperature in Kelvin, and
Vm is the test membrane potential.
Because the apparent dissociation constant for RuR was measured at +20
mV (taken as the IC50), the
KD20 must be used;
this is accounted for in calculating the electric distance by
subtracting 20 mV from Vm inside the
exponential term. The final equation used was: fb = [1 + (KD20/[RuR]) · exp(z · (Vm 20) · F/RT)]1.
Single Channel Recording.
For recording single class C
channel activity, the oocyte vitelline membrane was removed by first
shrinking the cell in a hyperosmotic solution and then stripping the
vitelline membrane away using forceps. Stripped oocytes in the
recording chamber were bathed in a depolarizing solution (in mM: 100 KCl, 10 HEPES, 10 EGTA, pH 7.4 with KOH) to clamp the intracellular
potential at 0 mV. Patch pipets were pulled from borosilicate glass,
coated with Sylgard (Dow Corning, Midland, MI), and heat-polished to a
resistance of ~18 to 25 M when filled with the 110 mM
Ba2+ recording solution (in mM: 110 BaCl2, 10 HEPES, pH 7.4). In contrast to the 40 mM Ba2+ solution, there was no
Ba2+ precipitation in the 110 mM
Ba2+ solution. Cell-attached patches with seal
resistances of typically ~20 to 100 G were obtained, and single
channel currents recorded with an Axopatch 200B amplifier (Axon
Instruments, Foster City, CA), filtered at a corner frequency of 2 or 5 KHz (8-pole Bessel filter, Frequency Devices, Haverhill, MA), and
sampled at 10 or 25 KHz using Pulse (Instrutech Corp., Great Neck, NY)
software. The internal filter of the Axopatch 200B amplifier was set at 10 KHz in our experiments, so the effective corner frequency of the
cascaded filters was either 1.96 or 4.47 KHz. All single channel currents were inward currents carried by Ba2+. In
the single channel block experiments, RuR was included in the 110 mM
Ba2+ pipet solution. The trans-patch
membrane potentials described are conventional for cell-attached patch
recording, intracellular potential minus pipet potential. A
nondihydropyridine agonist, FPL 64176 (2 µM, RBI, Natick MA), was
included in the bath solutions to prolong channel open times and
thereby facilitate the investigation of putative open-channel pore
block. Analysis of single channel kinetics was carried out as described
previously (Lansman et al., 1986).
). Because block by RuR
was attenuated upon prolonged exposure of RuR-containing solutions to
light, RuR solutions were protected from light and made fresh daily.
All other chemical reagents were obtained from Fluka, Sigma, or Aldrich
Chemical Company (Milwaukee, WI). All experiments were conducted at
room (~22°C) temperature.
All mean values are reported together with S.E.M. Theoretical
relationships (Hill equation, Boltzmann functions, and
voltagedependence of block) were fit to the data using a nonlinear
least-squares routine (Marquardt-Levenberg algorithm) in SigmaPlot
(Jandel Scientific, San Rafael, CA).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Concentration Dependence of RuR Action.
To estimate the
binding affinity of RuR for various voltage-gated
Ca2+ channels, we studied the concentration
dependence of RuR block of these channels. Superimposed records in Fig.
1 illustrate the action of RuR on class
A, B, C, and E Ca2+ channels. Three
micromolar RuR blocked all four of the channel types we tested,
but to differing degrees. Class A and B channels were most sensitive to
block by RuR, class C channels were intermediate in sensitivity, and
class E channels were little affected by 3 µM RuR. Complete
dose-inhibition relations for block by RuR of these four classes of
voltage-gated Ca2+ channels are presented in Fig.
2. Block of class C and E channels was
well-fit by 1:1 binding functions, yielding IC50
values of 25.4 and 67.1 µM, respectively. The dose-inhibition
relationships for class A and B channels were very similar to one
another, and complex: one component (fraction = 0.75) of RuR block
was described by a Hill coefficient of nH = 2
and an IC50 of 0.7 µM, and a second component
(fraction = 0.25) could be well-fit using a Hill coefficient of
nH = 1 and an IC50 of
25.4 µM. Thus, class A and B channels can be blocked by RuR in two
distinct ways, whereas class C and E channels possess a single binding
site for RuR. Interestingly, the lower affinity component of block of
either class A or B channels was not distinguishably different from
block of class C channels.
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Voltage Dependence of RuR Action.
To further characterize the
mechanism of RuR block of voltage-gated Ca2+
channels, we examined the voltage dependence of RuR block in an attempt
to determine whether RuR blocks by binding in the channel pore, or
alternatively, if block results from modification by RuR of channel
gating. The effect of RuR upon current-voltage relationships for each
of the four Ca2+ channel types tested is
illustrated in Fig. 3A. Using doses of RuR that blocked between one- and two-thirds of the
Ba2+ current, we observed no significant change
in reversal potential for any of the four channel types studied (see
Legend for mean values). Furthermore, block by subsaturating doses of
RuR had little effect on the shape of the current-voltage relations,
for either the inward current carried by Ba2+ or
the outward current carried by K+. Figure 3B
plots fractional block of peak Ba2+ current
versus membrane potential, and shows that there was little voltage
dependence to RuR block of class A, B, or C channels, but that block of
class E channels was mildly, but clearly, voltage-dependent.
|
). The value of z was estimated by fitting a
modified form of the Boltzmann equation (see Materials and
Methods) to the data for class E channels in Fig. 3B (dashed
line), yielding a z of 0.36. Based on this potential mechanism of
RuR block, and assuming that z = +6, the binding site for the
blocker is very superficial ( = 0.06), located only 6% of the way
across the membrane electric field. However, RuR has a linear structure
of
[(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]+6
with the positive charge distributed evenly across the
Ru(NH3)n (n = 4 or 5) moieties. This extended structure is not expected to allow
the entire molecule to penetrate the electric field, so the effective
charge of the ion at the binding site is likely to be less than +6,
which in turn would place the binding site deeper in the electric field.
RuR might alternatively or additionally affect the gating of
Ca2+ channels. We therefore measured the voltage
dependence of channel activation for class A, B, C, and E channels in
zero RuR and in approximately half-blocking concentrations of RuR (Fig.
4A). Activation functions were calculated
from normalized conductance values obtained using a range of test
potentials because the slowness of the oocyte clamp precludes accurate
measurement of tail currents. The absence of significant voltage
dependence to RuR block of class A, B, or C Ca2+
channels is reflected in a similar lack of effect of RuR on activation functions for these three channel types (Fig. 4A). Likewise, the mild
voltage dependence of RuR block of class E channels is reflected in the
small positive shift in the apparent activation function for this
channel type. A positive (right) shift in the activation function is
consistent with the decrease in fractional block observed for
increasingly positive test voltages.
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Block of Closed Channels by RuR.
Can RuR block channels even
when the channel is not open? To help address this issue, we examined
whether RuR can block closed channels as well as it can block open
channels. Experiments such as that illustrated in Fig.
6 were specifically designed to test whether RuR block of nonactivated (unused) channels develops as rapidly
as when channels are regularly activated (used). In one set of
experiments, after establishment of a steady amplitude for peak inward
Ba2+ currents, RuR was applied and the time
course of block measured. In a separate set of experiments, after
establishment of a steady amplitude for peak inward
Ba2+ currents, the test depolarizations were
stopped and RuR was applied at the same time. After waiting 3 to 6 min,
the constant amplitude test depolarizations were restarted, allowing
measurement of Ba2+ currents and their fractional
block. Comparisons were made between different cells because the slow
reversibility of RuR block did not allow the two experimental protocols
to be carried out on the same oocyte.
|
Block of Single Open Channels by RuR. In attempting to determine whether RuR block of voltage-gated Ca2+ channels occurs fundamentally by modifying gating, or alternatively, by blocking the channel pore, we examined the action of RuR upon single Ca2+ channels. In these experiments, we studied the class C channel only, because openings can be prolonged in this channel type by the use of a channel agonist such as FPL 64176. Extended channel open duration is useful because it facilitates resolution of channel-blocking events.
Comparing single channel openings in the absence of RuR with those obtained in the presence of RuR reveals that the blocker causes brief interruptions in the flow of current through the class C channels (Fig. 7A). The longer shut times between the bursts of open/shut transitions correspond to the normal closed time of the channel in the absence of RuR. The number of brief blocking events increased with dose of RuR, such that the time-integrated current carried by an open channel is reduced by RuR. RuR solutions exposed to light produced fewer flicker block events, consistent with an interpretation of reduced block resulting from photodegradation of RuR dye (data not shown; see Materials and Methods).
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1
s1) is an order of magnitude below the
diffusion-limited encounter rate predicted for a molecule the size of
RuR, whereas the apparent dissociation rate (5849 s1) predicts that class C channels would
recover from block in less than 0.2 ms. The fast unblock of single
channels is in sharp contrast to the very slow unblock observed using
two-electrode voltage-clamp measurements. It is possible that the slow
recovery from block in two-electrode voltage-clamp experiments arose
from adsorption of RuR onto the oocyte membrane and that slow unblock
in those experiments in fact reflected slow removal of RuR from the
oocyte (Voelker and Smejtek, 1996). From single-channel kinetic
analysis, the apparent dissociation constant
(kdissociation/kassociation) is 344 µM, or about 13-fold larger than the
IC50 determined from the dose-inhibition data
(Fig. 2). Assuming that RuR must compete with
Ba2+ for entry into the channel mouth, the
discrepancy in apparent binding affinity is readily attributable to the
approximately 11-fold difference in Ba2+
concentration (see Materials and Methods) used in the two
kinds of experiments.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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RuR has a long and extensive history of use in many experimental
settings, including a well known role as an antagonist of one class of
intracellular Ca2+-release channels, the
ryanodine receptors. RuR has also been identified as a likely blocker
of voltage-gated Ca2+ channels in the cell
surface membrane (Stimers and Byerly, 1982; Tapia et al., 1985;
Hamilton and Lundy, 1995; Lin and Lin-Shiau, 1996; Tapia and Velasco,
1997; Hirano et al., 1998). However, direct observation of RuR block of
mammalian neuronal Ca2+ channel currents has not
been described previously. Furthermore, there are several major
subtypes of voltage-gated Ca2+ channels in
mammalian neuronal surface membranes, including L-, N-, and P/Q-type
channels, and the possible selectivity of RuR block of the various
subtypes has previously been unclear. Here we have shown, using
essentially pure populations of heterologously expressed channels, that
RuR blocks several kinds of mammalian voltage-gated
Ca2+ channels with half-block concentrations in
the range of those described previously for Ca2+
channels in native tissues. In contrast with some earlier reports, we
have found that L-type channels as well as non-L-type channels are
blocked by RuR. The results of our experiments also help to elucidate
the mechanism of RuR action upon these channels.
RuR Sensitivity of Distinct Channel Types. Both dihydropyridine-sensitive L-type Ca2+ channels (class C) and non-L-type channels (classes A, B, E) are sensitive to RuR. Previous studies, lacking the advantage of using nearly pure populations of channels, had come to conflicting conclusions in this regard, some suggesting that L-type channels are not sensitive to RuR. Part of the explanation for the discrepancy may be that class A and B channels are more sensitive to RuR than are class C channels. The results of our dose-inhibition measurements also address the possibility, raised by others in earlier work, that RuR might prove to be an inexpensive and readily available antagonist for particular Ca2+ channel subtypes. Based on our results, the differential sensitivity to RuR between channels involved in neurotransmission (classes A and B) and those that play other roles in cells (for example, class C), may be exploitable in a limited way, but the dose-inhibition relationships are not widely separated, which ultimately disfavors use of RuR as a means to discriminate among Ca2+ channel subtypes in cells possessing mixed populations of these channels.
Earlier work focused on functionally defined channel types present in native tissues, particularly P/Q-type (class A), N-type (class B), and L-type (class C) channels. Class E channels do not have a clear functional correlate, although they are often linked to "R-type" channels. Our characterization of RuR action on class E channels therefore represents the first description of such action. Class E channels were the least sensitive to RuR of the four channel types we tested. Class A, B, and E channels are closely related to one another and are structurally divergent from the non-L channels, including class C channels. Block of class A channels is virtually identical with block of class B channels, both in the multi-component nature of the dose-inhibition relationships and in the IC50 values. Why class E channels are different from the two other non-L-type channels, being less sensitive to RuR than are class C channels, is not clear. Inspection of pore-lining sequences, particularly in regions known to form the pore entrance, reveal differences in amino acid sequences, but little that could obviously account for the observed differences in RuR binding.Mechanism of RuR Block: Number of Sites. Block of class C and E channels is well described by binding of a single molecule of RuR per channel. The RuR binding site differs slightly in affinity between these two channel types, based on IC50 values of 25.4 µM for class C channels and 67.1 µM for class E channels. The difference in affinities translates to a free energy difference in the binding of RuR to these sites of about 0.6 kcal/mol, which is roughly one-tenth of the molar energy of hydrogen bonding.
Class A and B channels are very similar to one another in their block by RuR, but unlike C and E channels, there are two components to block of A and B channels. In our experiments >95% of the Ba2+ current is carried by the heterologously expressed channels, so a contaminating contribution of endogenous oocyte Ca2+ channels to the dose-inhibition relationships cannot account for the complex form of the data. The lower-affinity component, which comprises approximately 25% of block, can be described by the binding of one RuR molecule per channel. To the extent that stoichiometries and affinities are similar, the lower-affinity site on A and B channels may correspond to the single site of C and E channels. The higher-affinity component of block of A and B channels, which comprises approximately 75% of the total block of these channels by RuR, appears to involve the binding of two RuR molecules per channel (Hill coefficient is2). Full block by RuR of A
or B channels may require the binding of as many as three RuR molecules.
Comparison of RuR block of A and B channels to RuR block of ryanodine
receptors is useful. Hill slopes for block of ryanodine receptors by
RuR are 
2, the interpretation in that case being that two or more RuR
molecules bind within the pore of ryanodine receptors (Ma, 1993). If a
similar process occurs in A and B channels, then an additional
assumption must be made: even when the high-affinity sites are fully
occupied, the channel is not completely blocked (~75%).
Alternatively, this component of block may result from alteration by
RuR of channel gating.
Mechanism of RuR Block: Pore Blocker Versus Gating Modifier.
We have considered two mechanisms for RuR block of voltage-gated
Ca2+ channels: physical obstruction of the pore
or a shift in the voltage-dependent probability of channel opening.
Other mechanisms of block are less likely. Screening of membrane
surface charge by RuR is not the exclusive mechanism of block because
RuR blocks single channel currents in high ionic strength solutions
(110 mM Ba2+; Kuo and Hess, 1992; Block et al.,
1998). Loss of channels into a long-lived nonconductive state is
unlikely because single channels open very frequently even in the
presence of high concentrations (1 mM) of RuR.
1) and corresponds to the normal, slow
opening rate in the absence of RuR. The fast reopening rate describes
the return to the open state from the brief, RuR-induced shut events
(kreopen = 1/[brief shut lifetime] ~ 1000 s1). Such a mechanism is complex and
consequently somewhat less plausible than the relatively
straightforward pore block hypothesis.
An experimental paradigm that would be helpful in distinguishing
between the two block mechanisms involves comparison of RuR block for
two different concentrations of permeant ion on the opposite
(intracellular) side of the membrane. Testing for "knock-off" of
RuR by an internal permeant ion was not possible because
Ca2+ channels do not often survive patch
excision, and our attempts to borrow a strategy of elevating internal
K+ by injection into oocytes (Wang et al., 1996a)
failed to be useful for us because K+ injection
induced large outward currents that obscured any potential effects of
elevated K+ on RuR block of
Ca2+ channels. Until such experiments are done,
perhaps using whole-cell patch clamp and a heterologous expression
system, the pore block mechanism remains the likeliest model for RuR
action on voltage-gated Ca2+ channels. It should
also be emphasized that for class A and B channels, which apparently
have multiple RuR binding sites, multiple mechanisms of RuR block may
be involved.
| |
Acknowledgments |
|---|
We thank Lisa Siconolfi for help with preliminary experiments. We also thank Tsutomu Tanabe, Yasuo Mori, Franz Hofmann, and Veit Flockerzi for the generous gifts of calcium channel cDNAs.
| |
Footnotes |
|---|
Accepted for publication February 11, 1999.
Received for publication December 10, 1998.
1 This work was supported by National Institutes of Health Grants AG04418 and NS35245, and the American Federation for Aging Research Grant A96079.
Send reprint requests to: Dr. William A. Sather, Neuroscience Center, B-138, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. E-mail: william.sather{at}uchsc.edu
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Abbreviations |
|---|
RuR, ruthenium red.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) and during (
)
treatment with RuR (1 µM for class A, B; 10 µM for class C; 100 µM for class E). Average reversal potentials obtained from three to
five cells for each of the four classes of channels studied were
(mean ± S.E.M.): 69.1 ± 3.1 and 69.6 ± 4.7 mV for
class A (n = 3), 64.8 ± 4.0 and 64.9 ± 4.1 mV for class B (n = 4), 68.2 ± 1.6 and
71.2 ± 1.5 for class C (n = 5), and 75.8 ± 1.3 and 78.4 ± 2.2 mV for class E (n = 5)
before and during RuR treatment. The current-voltage relationships
peaked at (mean ± S.E.M.): 17.0 ± 1.5 and 17.7 ± 0.5 mV for class A (n = 3), 14.3 ± 3.0 and
17.3 ± 2.2 mV for class B (n = 4), 19.0 ± 0.9 and 20.8 ± 1.0 mV for class C (n = 5),
and 20.1 ± 1.1 and 25.6 ± 1.0 mV for class E
(n = 5) before and during RuR treatment. Currents
were recorded using step depolarizations from a holding potential of
