Békésy Laboratory of Neurobiology and Department of Zoology, University of Hawaii, Honolulu, Hawaii 96822
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ABSTRACT |
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Duan, Shumin and
Ian M. Cooke.
Selective inhibition of transient K+ current by
La3+ in crab peptide-secretory neurons. Although
divalent cations and lanthides are well-known inhibitors of
voltage-dependent Ca2+ currents
(ICa), their ability to selectively inhibit a
voltage-gated K+ current is less widely documented. We
report that La3+ inhibits the transient K+
current (IA) of crab (Cardisoma
carnifex) neurosecretory cells at ED50 ~5 µM,
similar to that blocking ICa, without effecting the delayed rectifier K+ current
(IK). Neurons were dissociated from the major
crustacean neuroendocrine system, the X-organ-sinus gland, plated in
defined medium, and recorded by whole cell patch clamp after 1-2 days in culture. The bath saline included 0.5 µM TTX and 0.5 mM
CdCl2 to eliminate inward currents. Responses to
depolarizing steps from a holding potential of 40 mV represented
primarily IK. They were unchanged by
La3+ up to 500 µM. Currents from
80 mV in the presence
of 20 mM TEA were shown to represent primarily
IA. La3+ (with TEA) reduced
IA and maximum conductance
(GA) by ~10% for 1 µM and another 10% each
in 10 and 100 µM La3+. Normalized
GA-V curves were well fit with a
single Boltzmann function, with V1/2 +4 mV and
slope 15 mV in control; V1/2 was successively
~15 mV depolarized and slope increased ~2 mV for each of these
La3+ concentrations. Cd2+ (1 mM),
Zn2+ (200 µM), and Pb2+ (100 µM) or removal
of saline Mg2+ (26 mM) had little or no effect on
IA. Steady-state inactivation showed similar
right shifts (from V1/2
39 mV) and slope
increases (from 2.5 mV) in 10 and 100 µM La3+. Time to
peak IA was slowed in 10 and 100 µM
La3+, whereas curves of normalized time constants of
initial decay from peak IA versus
Vc were right-shifted successively ~15 mV for
the three La3+ concentrations. The observations were fitted
by a Woodhull-type model postulating a La3+-selective site
that lies 0.26-0.34 of the distance across the membrane electric
field, and both block of K+ movement and interaction with
voltage-gating mechanisms; block can be relieved by depolarization
and/or outward current. The observation of selective inhibition of
IA by micromolar La3+ raises
concerns about its use in studies of ICa to
evaluate contamination by outward current.
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INTRODUCTION |
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Lanthanum chloride is widely used as a potent blocker of Ca2+ permeation through membranes. It is effective at micromolar concentrations when added to physiological salines having their usual concentrations of divalent cations. It is used to completely block calcium currents (ICa) to evaluate whether any voltage-dependent outward currents contaminate the currents recorded under regimes designed to isolate ICa and thus improve the characterization of voltage-dependent ICa. We found that La3+ inhibits the voltage-dependent, rapidly inactivating potassium current (IA) of crab secretory neurons at concentrations similar to those inhibiting ICa without affecting the delayed rectifier potassium current (IK).
Although inhibitory effects of divalent Ca2+-channel
blockers on both transient and delayed outward currents were described for a variety of invertebrate (e.g., Thompson 1977) and
vertebrate neurons (e.g., Mayer and Sugiyama 1988
),
there appear to be few reports of selective effects of La3+
on IA at concentrations normally used to block
ICa. Because negative results are rarely
reported, it is difficult to know how widely the effects of low
concentrations of La3+ on potassium currents have been
examined. Selective effects of La3+ on the
IA component of outward current of rat
cerebellar granule cells (Watkins and Mathie 1994
) and
hippocampal neurons (Talukder and Harrison 1995
) point
to the possibility that such actions may be more general than is
currently appreciated. Except for an abstract (Duan and
Cooke 1997
), the effects of La3+ on potassium
currents of crustacean neurons have not to our knowledge been
previously reported.
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METHODS |
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Dissection and culturing
The procedures used to dissociate and culture X-organ neurons
from the semiterrestrial tropical crab, Cardisoma carnifex
Herbst, were described in detail elsewhere (Cooke et al.
1989; Grau and Cooke 1992
). Briefly, the X-organ
with <1 mm of the axon tract was removed from the eye stalk of adult
male crabs and agitated in the dark for 1.5 h in a
Ca2+/Mg2+-free saline containing 0.1% trypsin
(Gibco). A large volume of Ca2+/Mg2+-free
saline was then added to retard enzymatic activity, and the cells were
dissociated by gentle trituration in a 60-µl drop of culture medium
on 35-mm Primaria dishes (Falcon 3801). The dishes were carefully
flooded after allowing 1-2 h for the cells to adhere to the
substratum. The culture medium consisted of Leibowitz L-15 (Gibco)
diluted 1:1 with double-strength crab saline to which D-glucose (120 mM, Fisher), L-glutamine (2 mM,
Sigma), and gentamicin (50 mg/ml, Gibco) were added. Cultures were
maintained in humidified incubators (Billups-Rothenberg) in the dark at
22-24°C.
Experiments were performed on cultured X-organ somata that were 2-3
days in culture. Cells whose regenerative outgrowth took the form of
large, lamellipodial growth cones ("veilers") (Grau and
Cooke 1992) and were thus identifiable as containing crustacean hyperglycemic hormone (Keller et al. 1995
) were chosen
for this study. Before starting electrophysiological recording, the
culture dish was rinsed three times, and the medium was replaced with filtered crab saline consisting of (in mM) 440 NaCl, 11 KCl, 13.3 CaCl2, 26 MgCl2, 26 Na2SO4, and 10 HEPES, pH 7.4 with NaOH. During the experiments, the dish was constantly superfused with crab saline at
a rate of ~0.2 ml/min. A self-priming siphon maintained a relatively
constant fluid level. Somata were viewed with a Nikon Diaphot inverted
microscope with a ×40 objective and Hoffman modulation optics.
La3+ application
The use of a miniature Y-tube (Murase et al.
1989) manipulated to within <25 µm of the soma permitted
rapid (<100 ms) application of La3+-containing saline to
the neuron with minimal dilution or mixing during patch clamping. The
solutions to be applied, held in reservoirs higher than the recording
bath, are drawn through the arms of the Y, which are of larger diameter
tubing (PE 50) than the stem (PE10), by gentle suction; some bath fluid
is drawn in through the stem ensuring that no agent is applied
unintentionally. A solenoid pinch valve controlled by a Grass S-15
stimulator through which the arm on the suction side of the Y passes is
used to stop the suction for a selected duration, permitting gravity
flow of the material out of the Y while suction is blocked.
Electrophysiology
Voltage-clamp recordings were obtained in the whole cell
patch-clamp configuration with an EPC9 amplifier (Instrutech, NY). Data
acquisition, storage, and analysis were performed with HEKA software
(Instrutech, NY) run on a Macintosh Centris 650 computer. Correction
for leak was achieved with a p/N of four scaled subtractions obtained
at zero-current hyperpolarizing command potentials. Signals were
filtered with a corner frequency of 2.9 kHz. All experiments were
recorded at room temperature (22-26°C). Pipettes used to obtain
tight-seal whole cell recordings were pulled from Kimax thin-walled
glass capillaries (1.5-1.8 mm OD) on a vertical puller (David Kopf
Instruments, TW 150F-4). Pipettes were coated with dental wax to reduce
capacitance and fire polished with a microforge (Narishige, model
MF-83). Pipettes filled with the intracellular solution and immersed in
the bath had resistances ranging from 1.5 to 5 M but typically 1.5 to 3 M
. The intracellular solution, unless otherwise noted, was (in
mM) 300 KCl, 10 NaCl, 5 Mg-ATP, 5 BAPTA, and 50 HEPES, pH adjusted to
7.4 with KOH. The extracellular solution was crab saline as described
previously with 120 mM D-glucose and with 0.5 µM TTX and
0.5 mM CdCl2 to suppress inward currents. The tonicity was
adjusted with sucrose to 1,095-1,100 mosm for intracellular solutions
and 1,100 mosm for extracellular solutions. After establishing an
electrode seal and breaking in, neurons were held at
50 mV, a value
close to the usual resting potential and therefore requiring very
little current, when recordings were not being made. Series resistance
(Rs), was <6 M
, typically ~3 M
; it was
compensated to the maximum possible without causing oscillation,
ringing. or overshoot. Larger cells (capacitance >50 pF) could be more
completely compensated and were compensated by 85-90%. The few
smaller cells recorded could be less fully compensated but were never
compensated at <60%. These cells also had smaller currents. The data
were not corrected for uncompensated Rs; we
estimate that errors in reported Vm caused by
Rs are <5 mV. To record voltage-gated currents
the potential was stepped from
50 mV to the intended holding
potential (Vh) for 300 ms before initiating
additional voltage commands.
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RESULTS |
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Isolation of IA and IK
In this study, outward currents were examined with a bathing
saline containing 0.5 µM TTX and 0.5-1 mM Cd2+ to
eliminate possible competing inward Na+ and
Ca2+ currents (Meyers et al. 1992;
Richmond et al. 1995
). The presence of TTX increased the
size of the initial outward current, whereas addition of
Cd2+ to TTX-containing saline decreased a dip in the
current traces between the early peak and later outward current but did
not alter the peak (<15 ms); the amplitude of the late outward current
(measured at 90 ms) was reduced by the presence of Cd2+ but
by <10% (data not shown) as also noted by Meyers et al. (1992)
.
As previously described for the cultured C. carnifex X-organ
neurons (Meyers et al. 1992), outward K+
currents having typical characteristics of IA
and IK can be readily isolated. Currents
representing a Ca2+-activated potassium current were not
resolved and if present were very small. The characteristics and
current densities of IA and
IK did not differ systematically among X-organ
neurons having different morphologies of outgrowth in culture nor with time in culture (Meyers et al. 1992
). Exploiting the
rapid inactivation of IA during depolarization,
currents in response to incremented depolarizing voltage commands were
recorded from a holding potential (Vh) of
80
mV, at which IA is fully available for
activation (Fig. 1A), and
again from Vh
40 mV (Fig. 1B), at
which IA is almost fully inactivated, leaving
responses primarily representing IK. The
responses at corresponding commands from Vh
40
mV were subtracted from those at
80 mV to provide estimates of
IA (Fig. 1E).
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Because La3+ causes voltage shifts of
IA activation and inactivation as detailed
subsequently, the use of different holding potentials to isolate the
currents was impractical. We thus evaluated the effectiveness, compared
with the use of different Vh, of using pharmacological blockers to separate IK and
IA (Thompson 1977). As for other
neurons, 4-aminopyridine (4-AP, 5 mM) effectively and relatively
selectively blocked IA (Fig. 1C),
whereas TEA (20 mM in the bath) selectively blocked
IK (Fig. 1D) and revealed transient
outward currents closely matching those obtained by subtraction (Fig.
1E). The slightly smaller currents seen in Fig. 1E compared with Fig. 1D reflect the presence of
IA remaining in responses from
Vh
40 mV (Fig. 1B). These can be
evaluated by comparing Fig. 1B, in which a small initial
hump is present in the records, with Fig. 1C in which 4-AP
has more completely blocked IA.
It will be noted that the currents we attribute as primarily
representing IA, whether observed as the result
of subtraction of currents obtained from Vh 40
from those at Vh
80 mV or from Vh
80 in the presence of 20 mM TEA, do not
completely inactivate over the duration of depolarizations lasting
>100 ms. Whether this residual current, consistently amounting to
~20% of peak IA, represents ion movement
through channels responsible for IA or for
IK remains undetermined. We have not corrected
measurements of IA for this noninactivating
component of current.
Unless otherwise noted, the observations on outward current presented subsequently were obtained in the presence of 0.5 µM TTX, 0.5-1 mM Cd2+, and 20 mM TEA. Under these conditions inward current was not apparent; voltage-dependent outward current will be referred to as IA.
La3+ inhibits IA but not IK
Figure 2 shows records of outward
currents in saline with TTX and Cd2+, but without TEA
(A) and after the addition of 100 µM La3+
(B) recorded from Vh 80 mV and
therefore eliciting both IA and IK (left panels) and from
Vh
40, primarily IK
(right panels). A neuron showing relatively large
IK was selected for this example. The early peak
evoked from Vh
80 mV representing
IA is absent in La3+, whereas the
late and sustained currents (here measured at ~90 ms) are unaffected.
In Fig. 2C, the measurements of current at 90 ms for control
saline from Vh
40 mV are plotted with the
observations in La3+ from both Vh
40 and
80 mV. The responses are almost superimposable. Concentrations of La3+ >0.5 mM were required to obtain a
noticeable effect on IK, seen as a right-shift
of the current-voltage relation (data not shown).
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Figure 3 presents typical observations on
IA current (isolated by recording in saline with
TTX, Cd2+, and TEA) from a veiling neuron exposed to a wide
range of La3+ concentrations. In Fig. 3A,
currents recorded in response to steps to +15 mV from
Vh 80 mV in control saline (a command evoking ~80% of the maximum response) and saline with 1, 10, and 100 µM La3+ are superimposed. The initial outward current peak is
reduced, and its activation and inactivation slowed with increasing
[La3+]; in 100 µM no peak is seen in response to the
+15 mV command, and there is only a non- or slowly inactivating
residual current. With more depolarizing commands peaks are observed,
as reflected in plots of the conductance giving rise to
IA, GA versus
Vc (Fig. 3, B and C). As
mentioned previously, the responses in La3+ at modest
Vc lacking an initial peak may represent
IK that is not completely blocked by TEA. Figure
3B presents plots of specific conductance (see figure
legend) versus command voltage (Vc) for the same
neuron (symbols). The data were fitted with a single Boltzmann function
as given in the figure legend (Fig. 3B, lines).
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The major effects of the addition of La3+ can be summarized as follows.
1) As seen for the neuron providing the data of Fig. 3B, there is a reduction in the maximum IA current and conductance that could be obtained with depolarizing voltage commands. A summary of all the observations is shown in Fig. 4A, which plots the averaged observations of the reduction of maximum conductance by La3+ from eight neurons examined. The reduction relative to control saline is nearly linear when plotted against the log [La3+] and amounts to ~30% at 100 µM [La3+].
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2) There is a shift of ~15 mV to more depolarized voltages (a right shift) of corresponding points of the IA-V and GA-V curves in 1 µM La3+ and a further shift of this magnitude with each 10-fold increase of [La3+], as seen in Figs. 3, B and C, and 4B and further described subsequently.
Changes in current amplitude might result from a change in relative
permeability or driving force for permeant ions. Tail currents in
response to repolarizing commands to a series of voltages after
IA activation were therefore examined to test
whether La3+ altered the reversal potentials.
La3+ did not appreciably alter the reversal potential of
nearly 80 mV, and thus it can be concluded that it does not alter the
selectivity of the channel (data not shown). This value of the reversal
potential is in good agreement with the calculated
EK, thus supporting the identification of the
current as a K+ current.
Cd2+ (1 mM) had no effect on IA, whereas Zn2+ (200 µM) and Pb2+ (100 µM) produced small (<10 mV) right shifts of the I-V curves. Removal of the 26 mM Mg2+ present in the normal saline had no effect (data not shown).
La3+ effects on IA activation
To further characterize the effect of La3+ in right shifting the voltage dependence of IA activation, the responses of different neurons tested in control and the [La3+] were normalized with respect to the maximum conductance observed for the particular neuron in each condition. The observations for the normalized GA-V curves averaged at each Vc (n = 7-8) are shown in Fig. 3C (symbols, with error bars, ±SE). Each of the normalized curves was fitted with a single Boltzmann function (Fig. 3C, lines). The values of the Boltzmann parameters used to fit the averaged normalized GA-V curves are plotted against log [La3+] in Fig. 4B. The V1/2, a measure of the right shift of the voltage dependence of activation, increased approximately linearly with the log [La3+] from a control value of approximately +4 mV to approximately +49 mV in 100 µM La3+, ~15 mV per 10-fold increase in La3+. Voltage sensitivity was reduced by the presence of La3+ as indicated by an increase in the slope factor, from 15 mV in control saline to 22 mV in 100 µM La3+.
La3+ effects on IA steady-state inactivation
The effect of La3+ on the voltage dependence of
steady-state inactivation was examined with a double-pulse regime as
illustrated in Fig. 5. A 300-ms
hyperpolarizing or depolarizing prepulse (100 to +20 mV) from
Vh
70 mV was followed by a test depolarization chosen to evoke ~80% of the maximal IA, +20
mV for control saline, +30, +45, and + 60 mV for the 1, 10, and 100 µM La3+ salines. Previous studies (Meyers et al.
1992
) have shown that 300 ms is ~10 times the maximum
duration required to obtain the full extent of a change in steady-state
inactivation, regardless of the voltage. A selection of the
IA current traces from a typical experimental
series is shown in Fig. 5A. In control saline, the responses
after prepulses to
70 and
55 mV are superimposable while in saline
with 10 µM La3+ responses after prepulses to
55 and
40 mV are superimposable. Figure 5B plots the fraction of
the IA current in the absence of a prepulse over
that after a prepulse versus the prepulse voltage. The prepulses from
100 up to
55 mV were without effect on IA under any conditions, inactivation reached its maximum (i.e., IA was minimal) with prepulses to
10 mV in
control saline or 1 µM La3+-saline, and more depolarized
prepulses were required with increasing [La3+]. There was
consistently a residual outward current of ~20% that failed to
inactivate. The curves were fitted by a single Boltzmann function, and
the parameters are plotted in Fig. 5C. These indicate that,
although for 1 µM La3+ the curves were not shifted, the
voltage dependence of steady-state inactivation at 10 and 100 µM
La3+ is right shifted by ~15 mV for each 10-fold increase
in [La3+]. The slope is approximately doubled in 10 µM
La3+ relative to the control value but not increased by a
similar amount at 100 µM.
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La3+ effects on IA kinetics
Inspection of the IA responses (e.g., Fig. 3A) showed that addition of La3+ not only reduced the amplitude but slowed activation as reflected by broadened peaks. The time to peak decreased with increasingly depolarized commands and reached a minimum, typically ~3 ms, for depolarizations to more than +45 mV in normal saline. In the analysis shown in Fig. 6A to compare different neurons the time to peak for each voltage command is normalized relative to the asymptotic minimum in control saline at +60 mV. The average (n = 4-8) for each Vc for all the neurons is plotted versus Vc. Saline with 1 µM La3+ showed little effect on the time to peak, whereas higher [La3+] right shifted the curves. The failure to show a right shift of the plot of relative time to peak versus Vc in 1 µM La3+ could reflect that GA is maximal and reduced only ~10% in 1 µM La3+ at this Vc. With sufficiently large depolarization, the same asymptotic minimum time to peak as in control saline was obtainable in the La3+ salines.
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The rate of the fast, initial phase of inactivation that is
characteristic of IA is also slowed by the
presence of La3+. The voltage dependence of the rate of
inactivation was evaluated by fitting a single exponential to the
initial decline of IA responses to a series of
commands. To compare observations from the several neurons, the values
of the time constants at each Vc were normalized with respect to the asymptotic minimum observed for the neuron in
control saline (+90 mV). The averages of these values
(n = 4-8) are plotted versus Vc
in Fig. 6B for the control saline and the three
[La3+]. This analysis shows a right shift of the curves
with increasing [La3+]. The Vc at
which is doubled is shifted ~20, 40, and 60 mV more positive for
1, 10, and 100 µM La3+ respectively.
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DISCUSSION |
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This study has shown that La3+ at micromolar
concentrations, in addition to its well-recognized ability to block
membrane Ca2+ conductances, inhibits the rapidly
inactivating K+ current (IA) of crab
secretory neurons. The inhibition of IA is
selective, as mM concentrations are needed to observe effects on the
delayed rectifier K+ current. The [La3+]
needed for half-maximal blocking of IA is
similar to that for blocking the calcium current
(ICa) of these neurons and is between 1 and 10 µM. La3+ is the most effective known blocker for the crab
ICa, with Cd2+ having nearly equal
potency (Richmond et al. 1995). La3+
produces a concentration- and voltage-dependent block of
IA; the maximum conductance and current that can
be elicited is reduced, and the current- and conductance-voltage
relations are shifted to more depolarized values.
The plot of normalized time constants of the decay from peak
IA versus Vc shows a
depolarizing shift with increasing [La3+] that is also
similar to that for the G-V relations (~15
mV/10 × increase in [La3+]). This is consonant with
observations linking fast, N-type inactivation to activation
(Hoshi et al. 1991; Zagotta and Aldrich
1990
). Steady-state inactivation, as observed here with 300-ms
prepulses, may also be attributed to N-type, as contrasted with C-type,
inactivation. Thus a right shift of the fractional steady-state
inactivation of IA versus prepulse voltage would
be expected, as is observed. The lack of a change for the
observations in 1 µM La3+ may be attributable to the
choice of Vc for the test pulse, the more
depolarized value used in the La3+ saline (+30 rather than
+20) having compensated for the ~15 mV right shift of the
G-V curve relative to control saline for 1 µM
La3+.
The prepulse regimes for analysis of steady-state inactivation show a consistent 20% of the outward current that remains under all conditions. This corresponds to the current that is seen to remain at the end of long (>100 ms) depolarizations, whether IA is obtained by subtraction of records at different Vh or recorded after blocking IK with TEA (Fig. 1), as for the data considered in this paper. It is unclear whether this should be regarded as representing reactivation of conductance through IA channels or current through IK or other channels, a question that would require single-channel recording to resolve. Although this residual conductance was included as a parameter in fitting the Boltzmann parameters to data for steady-state inactivation, no other correction for this possible contamination of IA with other outward current was made in the analysis.
Our conceptual model of La3+ interaction with the IA channel visualizes repetitive binding and unbinding of a La3+ ion at a site part way into the pore channel. The right shift of voltage dependence as well as the voltage-dependent block of current is suggested to result from interactions with gating mechanisms when La3+ is bound at a specific site and obstruction of the pore by the La3+ when present in the channel. The ability of large depolarizing clamps to relieve block might result from the pulse expelling the blocking ion from the pore or the increased electrical driving force on K+ permitting it to dislodge La3+ as it moves outward through the pore.
Such a model is closely analogous to that developed by Woodhull (1973)
for block of conductance in Na+ channels by protons. The
assumptions made for development of that model appear reasonable for
the case of La3+ block of the IA
channel (for an application of this model to La3+ block of
a barley-root voltage-dependent K+ current see
Wegner et al. 1994
). Figure
7 presents fits to a Woodhull model of
the data used for Fig. 3. The fit to the observations are best for 1 µM La3+ and become poorer with increasing
[La3+], especially for modest depolarizing commands. The
Woodhull model provides estimates of the apparent affinity of the pore
binding site in the absence of a voltage across the membrane and the
distance across the electric field of the membrane of the binding site for different concentrations of the blocking ion. The affinities ranged
from 1 to 8 µM for the 1- to 100-µM range of [La3+]
examined, and distances varied from 0.2 to 0.34 of the membrane field
(Fig. 7 legend).
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In Woodhull's (1973) observations on [H+] effects on
Na+ currents, right shift of the voltage dependence was
separated from H+ block and was attributed to change of
surface charge. However, an effect of La3+ on surface
charge (Latorre et al. 1992
) seems unlikely for the crab
neurons; La3+ was effective at micromolar concentration in
the presence of a high ionic strength saline and high concentrations of
both Ca2+ (13 mM) and Mg2+ (26 mM). Rather we
suggest that changes of the voltage dependence and voltage sensitivity
of IA conductance result from an electrostatic interaction between gating charges and the ion when bound to a selective site near or in the pore. This explanation was proposed in a
number of studies of effects of La3+ and/or of divalent
cations on voltage-gated channels. These include observations in
typical vertebrate salines (e.g., Spires and Begenisich 1994
; Talukder and Harrison 1995
) as well as of
squid axons in high ionic-strength saline (Armstrong and Cota
1990
; Gilly and Armstrong 1982a
,b
).
Although there appear to have been a number of studies comparing the
effects of divalents on different types of K+ currents (see
Mayer and Sugiyama 1988), relatively few examined the
effects of lanthides. Ours is the first full report on crustacean neurons to our knowledge. In cultured hippocampal rat neurons (Talukder and Harrison 1995
), La3+ differed
from the other ions having effects at less than millimolar concentrations, Pb3+, Gd3+, Cd2+,
and Zn2+, in its low threshold (~5 µM) and in producing
a marked reduction of maximum IA current in
addition to right shifting the activation and inactivation curves as
did the divalents. La3+ and the others, unlike
Zn2+, which caused a parallel right shift in the activation
curves, decreased the voltage sensitivity of the activation curves.
La3+ (and Pb3+), unlike Cd2+ and
Zn2+, also inhibited the delayed rectifier current
(IK), although at a much higher concentration
(100 µM) in these hippocampal neurons. We report here that unlike in
the hippocampal neurons Pb3+, Cd2+, and
Zn2+ had little effect on the crustacean neurons.
Marked effects of La3+ on K+ currents but much
less selectivity for IA was seen in a study of
rat cerebellar granule neurons (Watkins and Mathie
1994). In the cerebellar neurons a separation of
La3+ effects on activation and inactivation was observed,
with inactivation voltage relations being right shifted more than
activation so that with appropriate pulse regimes enhanced
IA currents could be elicited. In both cultured
rat superior cervical ganglion neurons and embryonic chick sympathetic
neurons, 1 µM La3+ was seen to enhance transient outward
current (Przywara et al. 1992
). Although the
I-V curves appeared to have been shifted to more
polarized voltages, data on prepulse effects on steady-state inactivation were not presented. Thus the enhanced
IA may represent the result of a depolarizing
shift of the voltage dependence of steady-state inactivation.
Finding apparently different effects of divalents or lanthides on
the voltage dependence of activation and fast inactivation raises the
question of how independent these processes are. Values of the
V1/2 and slope for activation and steady-state
inactivation differ in all preparations examined, with the value of
V1/2 usually more polarized and the slope factor
smaller (voltage sensitivity greater) for inactivation. As briefly
reviewed previously, although some of the di- and trivalent cations
produce parallel shifts of activation or inactivation
I-V curves, in several there are changes in the
slope factor as well. The extent of the shifts and of changes in slope
factor may differ for activation and steady-state inactivation, as seen
in this study. In ours and the experiments discussed previously it was
not feasible to analyze the effects of possible rapid time-course
interaction between activation and inactivation. From analyses of
macroscopic and single-channel currents of a cloned
Drosophila shaker channel expressed in Xenopus oocytes, Zagotta and Aldrich (1990) concluded that inactivation occurs
from a transition closed state penultimate to opening. This analysis
thus links inactivation voltage relations and kinetics with those of
activation. A more complex scheme involving 15 closed states rather
than 5 in the path to opening was later used to model opening kinetics
of a mutated shaker A1 K+ channel with a
truncated N-terminus lacking fast (N-type) inactivation (Schoppa
and Sigworth 1998
; Zagotta et al. 1994
). A
similarly detailed model including inactivation has not yet been
published to our knowledge. The existence of several genes coding
transient K+ channels as well as a large number of splice
variants of these gene products provide a structural basis for finding
much variation in the biophysical characteristics of transient
K+ channels and the influence on them of trivalent and
divalent cations.
If La3+ blocks outward current at concentrations similar to those blocking ICa, its use to evaluate the presence of residual outward currents in studies of voltage-gated ICa is invalid. Our work suggests that it is important to also evaluate the effects of La3+ on outward currents in the absence of inward currents. It will be of interest to learn how general the selective inhibition of IA by La3+ may be.
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ACKNOWLEDGMENTS |
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We thank J. W. Labinia for preparing primary cultures of X-organ neurons as well as for unfailing technical assistance; Prof. Martin Rayner for critical comment on drafts of the mamnuscript; and Dr. Marc Rogers for helpful discussion.
This work was supported by the Cades Fund and by National Institute of Neurological Disorders and Stroke Grant NS-15453.
Present address of S. Duan: Neurology Service (VA-127), University of California, Veterans Administration Medical Center, 4150 Clement St., San Francisco, CA 94121.
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FOOTNOTES |
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Address for reprint requests: I. M. Cooke, Békésy Laboratory of Neurobiology, University of Hawaii, 1993 East-West Rd., Honolulu, HI 96822.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 23 October 1998; accepted in final form 29 December 1998.
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