1Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah Health Sciences Center, Salt Lake City, Utah 84132; and 2Bruce Rappaport Faculty of Medicine, Technion and Rappaport Institute, Haifa 31096, Israel
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ABSTRACT |
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Solessio, Eduardo, Kevin Rapp, Ido Perlman, and Eric M. Lasater. Spermine Mediates Inward Rectification in Potassium Channels of Turtle Retinal Müller Cells. J. Neurophysiol. 85: 1357-1367, 2001. Retinal Müller cells are highly permeable to potassium as a consequence of their intrinsic membrane properties. Therefore these cells are able to play an important role in maintaining potassium homeostasis in the vertebrate retina during light-induced neuronal activity. Polyamines and other factors present in Müller cells have the potential to modulate the rectifying properties of potassium channels and alter the Müller cells capacity to siphon potassium from the extracellular space. In this study, the properties of potassium currents in turtle Müller cells were investigated using whole cell voltage-clamp recordings from isolated cells. Overall, the currents were inwardly rectifying. Depolarization elicited an outward current characterized by a fast transient that slowly recovered to a steady level along a double exponential time course. On hyperpolarization the evoked inward current was characterized by an instantaneous onset (or step) followed by a slowly developing sustained inward current. The kinetics of the time-dependent components (block of the transient outward current and slowly developing inward current) were dependent on holding potential and changes in the intracellular levels of magnesium ions and polyamines. In contrast, the instantaneous inward and the sustained outward currents were ohmic in character and remained relatively unaltered with changes in holding potential and concentration of applied spermine (0.5-2 mM). Our data suggest that cellular regulation in vivo of polyamine levels can differentially alter specific aspects of potassium siphoning by Müller cells in the turtle retina by modulating potassium channel function.
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INTRODUCTION |
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Müller cells play an
important role in retinal physiology. They play a role in maintaining
retinal cell metabolism (Newman 1996; Reichenbach
et al. 1993
) and are essential for maintaining potassium
homeostasis of the extracellular space during neuronal activity
(Karwoski et al. 1989
; Newman
1987
; Newman et al. 1984
). To maintain
potassium homeostasis of the extracellular space, Müller cells
are highly permeable to potassium ions, predominantly through inward
rectifier potassium channels (Brew et al. 1986
; Chao et al. 1994
, 1997
; Newman
1987
, 1988
, 1993
; Nilius
and Reichenbach 1988
). Calcium-activated potassium
channels (Bringmann et al. 1997
; Newman
1985
; Puro et al. 1989
) and voltage-activated
potassium channels (Chao et al. 1994
; Reichelt
and Pannicke 1993
) have also been described but probably play
only a minor role in potassium homeostasis.
The biophysical properties of potassium channels in Müller cells
determine the efficacy with which these cells can maintain potassium
homeostasis. Recent studies demonstrated that Müller cells in
rabbit, rat, and mouse retina express Kir4.1 inward rectifier channels
(Ishii et al. 1997; Kofuji et al. 2000
).
Similar to other inward rectifier channels (Fakler et al.
1994
; Ficker et al. 1994
; Lopatin et al.
1994
), spermine plays a dominant role in conferring potassium
channels in rabbit Müller cells with voltage-dependent rectification (Biedermann et al. 1998
). Müller
cells have the capacity to produce polyamines such as spermine, which
are postulated to regulate potassium currents and modulate neuronal
function in vivo (Biedermann et al. 1998
). Thus the
degree of rectification may be modified by the intracellular milieu of
Müller cells and by intracellular metabolic pathways.
Previous studies (Conner et al. 1985; LeDain et
al. 1994
; Linn et al. 1998
) demonstrated that
turtle Müller cells are highly permeable to potassium ions and
that potassium fluxes could be induced in an inward or outward
direction dependent on the driving force. The outward currents
consisted of a fast onset transient followed by a sustained component.
At potentials more negative than the resting potential of the cell, the
currents had properties of inward rectifying channels. Like the
potassium channels in the endfeet of Müller cells in frog
(Skatchkov et al. 1995
), barium ions blocked both inward
and outward currents, cesium ions blocked only the inward current
(Linn et al. 1998
; Solessio et al. 2000
),
while tetraethylammonium (TEA) had no effect on either current
(Le Dain et al. 1994
; Linn et al. 1998
).
The stoichiometry of block by intracellular barium ions differed with
the state of polarization of the cells. The inward currents induced by
hyperpolarizations required two "apparent" binding sites for block
by barium ions, whereas a single apparent binding site was available
for block of the outward current during prolonged depolarizations. A
parsimonious interpretation of these findings suggested that on
depolarization, divalent ions and polyamines, known to block inward
rectifier channels, were driven into the channel pore, thereby
competing with the barium ions for binding sites (Solessio et
al. 2000
). It follows then, that, if the concentration of
intracellular polyamines is sufficient to completely block the
rectifying component, the inward and sustained outward currents most
likely flow through two different types of channels. Thus the nature of
the K+ channels present, their regulation by
polyamines, and their exact role in the siphoning of
K+ in turtle Müller cells remains unclear.
This is of particular importance in view of the complex structure of
turtle Müller cells (Conner et al. 1985
;
Linn et al. 1998
) that raises concerns about their
ability to siphon K+. To better understand the
physiology of turtle Müller cells, we undertook an investigation
of the mechanisms underlying the rectifying properties of the potassium
channels in turtle Müller cells and their role in potassium
siphoning. While spermine, and other polyamines, are effective
modulators of Müller cell potassium currents (Biedermann
et al. 1998
), we have limited knowledge of their role in the
overall physiology of the Müller cells. Our purpose here was to
characterize the different types of potassium currents, determine their
functional contribution to the overall membrane properties of the
cells, and evaluate the effects that up or down-regulation of
polyamines may have on the potassium current (and its particular
components). Applying whole cell techniques to freshly dissociated
cells, we recorded the membrane currents while controlling the
intracellular levels of divalent ions and polyamines such as spermine.
Our results demonstrate that two components contribute to the potassium currents in turtle Müller cells. A rectifying, time-dependent component that is blocked by spermine; and a second, approximately "ohmic" component, with little sensitivity to added spermine (0.5-2 mM). The findings described here and elsewhere are compatible with two alternatives: 1) the two current components are mediated by two different types of channels or 2) one type of inwardly rectifying potassium channel exists with a wide inner pore that can simultaneously accommodate more than one blocking ion and through which ions do not necessarily flow in single file.
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METHODS |
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Preparation
The study was conducted on Müller cells isolated from the retina of the fresh water turtle Pseudemys scripta elegans. Turtles were killed in accordance with the guidelines set out by the Association for Research in Vision and Ophthalmology for the use of animals in research. The eyes were enucleated and the globes then hemisected. The retinas from the two eyes were isolated from the eyecups and placed in a calcium/magnesium-free Ringer solution for 15 min. The isolated retinas were then incubated for 1/2 h in papain (100 U/20 ml) dissolved in L-15 (GIBCO BRL, Gaithersburg, MD). Then the retinal pieces were gently triturated onto glass coverslips. Recordings were performed within 4-10 h following dissociation.
Turtle Müller cells have five to six thin processes extending
from the cell soma, each ending in an endfoot (Conner et al. 1985; Linn et al. 1998
). This complex structure
prevents adequate space clamp when the whole cell recording
configuration is established in the soma of the cell (Perlman et
al. 2001
). We have found that the cell soma and the processes
of turtle Müller cells express potassium channels with similar
biophysical properties if at different densities (Perlman et al.
2001
). So we limited all recordings in this study to
Müller cells that had lost their processes during the isolation
procedures. Application of cable equations predicts that for cells
under these conditions, the space clamp is complete and allows accurate
determination of the biophysical properties of the different channels
underlying the currents measured (Perlman et al. 2001
).
Experimental solutions
The normal turtle Ringer consisted of the following (in mM): 110 NaCl, 2.0 CaCl2, 2.6 KCl, 2.0 MgCl2, 8.4 HEPES, and 10 D-glucose with pH 7.4. All chemicals were purchased from Sigma (St. Louis, MO).
Solutions were delivered via a 12-reservoir pressure ejection system
controlled by a personal computer (DAD-12, Adams, NY). The patch
pipette solution consisted of 130 mM potassium gluconate, 4 mM NaCl,
0.2 mM EGTA, 2 mM MgCl2, 10 mM HEPES, and 7.8 µM CaCl2 with pH 7.4. In some of the
experiments the pipette solution was supplemented with 500 µM
spermine. In other experiments we buffered spermine and other
polyamines by adding 5 mM tris-ATP (Watanabe et al.
1991) to a divalent-free solution. For the divalent-
(Ca2+ and Mg2+) free
solution, both MgCl2 and
CaCl2 were omitted, and EDTA and EGTA were added
at 5 mM each. Supplementing the intracellular solutions with EGTA,
EDTA, and/or ATP introduced less than a 5% change in osmolarity of the
intracellular medium.
Recording procedures
Whole cell recordings (Hamill et al. 1981)
were made with micropipettes that had been pulled on a two-stage puller
(model PP-83, Narishige Instruments, Tokyo, Japan) and were used
unpolished and coated with silicone elastomer (Sylgard). The electrode
tip resistance was 2-3 M
when measured in the bath solution. Series resistance and capacitance were compensated for electronically. Electrical potentials were recorded with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Data collection was controlled by
a personal computer interfaced to a Digidata 1200 data acquisition system driven by the pClamp suite of programs (Axon Instruments). After
establishing a whole cell configuration, the cells were held at a
potential (HP) of
80 mV unless indicated otherwise. The stimuli,
short (60 ms) or long (1 s or longer) incremental and decremental
voltage pulses, were preceded by a brief prepulse to
100 mV to
preactivate the channels.
Data analysis
Current-voltage functions (I-Vs) were measured from
the responses to incremental and decremental voltage pulses. The chord conductances were computed according to
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(1) |
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(2) |
Fits were performed with Sigma Plot (Chicago, IL), which applies the
Marquardt-Levenberg algorithm to minimize the sum of the squared
differences between the Boltzmann equation and the conductance data.
Tests for statistical significance were performed using ANOVA.
Conductance plots show average values measured at respective
potentials, bars are standard deviations, whereas number of
observations are indicated in the text. The time-dependent currents
were fit with two exponential curves
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(3) |
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RESULTS |
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Figure 1A shows the
current responses of an isolated cell soma to a series of 60-ms-long
voltage steps, applied from a holding potential of 80 mV. A prepulse
to
100 mV preceded each voltage step. Inward currents readily reach a
sustained level, as is characteristic of inward rectifiers (note that
the prestep to
100 mV fully preactivated this current). The outward
currents showed a fast initial transient followed by a slower
inactivation. Similar recordings were done from the same cell exposed
to different concentrations of extracellular potassium. The
corresponding I-V curves evaluated 25 ms following onset of
the voltage pulses (dashed line in Fig. 1A) showed only mild
rectification (Fig. 1B). A strong dependency on
extracellular potassium was revealed by the depolarizing shift of the
resting potential (46.2 ± 3.3 mV/decade, mean ± SD,
n = 4), an increase in the slope conductance (fit with
a power function with coefficient 0.7 ± 0.06, n = 4), and a crossover of the curves with increased concentrations of
extracellular potassium ions. The latter effects imply a rather weak
blocking mechanism underlying inward rectification that can be overcome
by an increase in the outward flow of potassium ions through the
channels (Hille and Schwarz 1978
).
|
Application of longer voltage pulses revealed an additional block that
developed at a slower rate (Fig. 1C). The current responses to long (1.6 s), depolarizing voltage pulses exhibited a decay of the
outward currents that could be described by the sum of two
exponentials. A fast component characterized by a time constant ranging
between 2 and 5 ms and a second slower component with a time constant
of about 100 ms (see Fig. 4D). The I-V curves were generated from data measured 1 s into the pulses (dashed vertical line in Fig. 1C) and are shown in Fig.
1D. As observed in response to the short (25 ms) pulses,
these curves showed a strong dependency on extracellular potassium
concentration. Raising extracellular potassium induced a depolarization
of the resting potential that was approximately Nernstian in nature
(47.5 ± 1.9 mV/decade, n = 4), suggesting a
selective conductance for potassium. This study replicated the work of
Newman (1993) in salamander Müller cells, and
these results are similar to his findings including the slope
conductance, which followed a power function with a power coefficient
of 0.68 ± 0.07 (n = 4). However, in contrast to
the I-Vs obtained when measured at 25 ms (Fig.
1B), these I-V curves showed strong rectification
and did not cross over as potassium levels were increased. This implies
an augmentation of the block with time that cannot be overcome in spite
of an increased potassium gradient (Hille and Schwarz
1978
).
To study the temporal progression of the block, we compared in Fig.
2A the I-V curves
computed from the same cell, at 4, 25, and 1,000 ms following the onset
of the voltage pulses. The curves overlapped at potential levels that
were hyperpolarized from the resting potential, indicating that the
inward currents developed within 4 ms or less and stabilized. At
depolarized levels the I-V curves departed considerably from
each other with rectification becoming more apparent with time.
However, even at the long time period (1 s) when a steady state was
achieved, the currents increased linearly with voltage, thus falling
short of complete rectification. A quantitative measure of the
rectifying properties of the channels in the Müller cells was
obtained by fitting the chord conductances with a Boltzmann function as
shown in Fig. 2B (Lopatin et al. 1995;
Newman 1993
). As rectification increased with time, the conductance curve sharpened. The Boltzmann coefficients needed to fit
the curves increased significantly (P < 0.01) from
1.25 ± 0.05 (n = 7) at 4 ms, to 1.5 ± 0.08 (n = 8) at 25 ms and 2.23 ± 0.19 (n = 9) at 1,000 ms. These values are typical of weak
rectifiers (Hille 1992
). Furthermore, there is a
concomitant decrease in the value of the pedestal needed to generate a
fit of the Boltzmann function. The standing conductance values
progressively decreased significantly (P < 0.01) from
0.53 ± 0.045 (n = 7), to 0.43 ± 0.01 (n = 8) and 0.28 ± 0.017 (n = 9)
for 4, 25, and 1,000 ms, respectively. A significantly more depolarized
blocking voltage (P < 0.01) was observed at 4 ms
(
57 ± 6 mV, n = 7), compared with those
determined at 25 (
74 ± 4 mV, n = 8) and 1,000 ms (
75.5 ± 5.7 mV, n = 9).
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Three distinct current features are apparent from the responses of
turtle Müller cells to voltage pulses (Fig. 1); an inwardly rectifying current, a transient outward current, and a sustained outward current. Although all three features are effectively blocked by
barium, none is sensitive to TEA or 4-aminopyridine (4-AP) (Le
Dain et al. 1994; Linn et al. 1998
;
Solessio et al. 2000
), which suggests that the outward
currents (transient and sustained) do not flow through typical
IA or
IK type potassium channels. However,
these currents are strongly affected by holding potential (Fig.
3). Holding the cells at a depolarized
membrane potential (
40 mV) results in a reduction of the transient
outward currents, and also a slowing down in the development of the
inward currents in response to hyperpolarizing pulses (Fig.
3A). These effects are readily observed in the
I-V curves that were measured 4 ms after onset of voltage
pulses (Fig. 3B). Both inward and outward currents are
smaller when the holding potential is set at
40 mV compared with
80
mV. The corresponding conductances undergo a significant
hyperpolarizing shift of the blocking voltage values to
139 ± 2.9 mV (P < 0.01, n = 5) compared with
57 ± 6 mV (n = 7) for a holding potential of
80 mV (Fig. 3C). There is also a reduction in the pedestal
level that has a value of 0.43 ± 0.05 (P < 0.01, n = 5) compared with 0.53 ± 0.045 (n = 7) at
80 mV; although the slope of the function
as inferred from the Boltzmann coefficients has not changed
significantly (1.16 ± 0.32, n = 5). Given that
the inward currents slowly relax to their standing levels, the effects
of holding potential are less pronounced with time after onset of the
test pulses. When measured at 25 ms (Fig. 3D), the blocking
voltage recovered to a hyperpolarized value of
96 ± 5.3 mV
(P < 0.01, n = 5), compared with
74 ± 4 mV (n = 8) at a holding potential of
80 mV. There was no significant change in the pedestal level
(0.39 ± 0.03, n = 5), and the estimated Boltzmann
coefficient of 1.94 ± 0.25 (P < 0.01, n = 5) reflects a sharpening of the block compared with
1.5 ± 0.08 (n = 8) obtained with a holding
potential of
80 mV. The effects of holding potential lasted <1 s as
no significant changes were detected when currents were measured 1 s after the onset of test pulses. The voltage-dependent changes that we
observed in the currents of turtle Müller cells were similar to
those that had been observed in recombinant Kir channels expressed in murine fibroblast cells (Ishihara
1997
). Here the holding potential levels that were depolarized
with respect to the reversal potential for potassium (in turtle
Müller cells this value is about
85 mV), promoted the
voltage-dependent block of the potassium channels by divalent ions and
polyamines.
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When Müller cells were studied for prolonged periods of time (4 min or more), we noticed that the degree of rectification and the rate
of its development changed with time, as shown in Fig.
4A. To investigate the outward
current, a depolarizing pulse to 0 mV was applied after the inward
rectifying channels were activated by a prepulse to 100 mV. The
outward current rose to a transient peak and then recovered gradually
to a steady state. Four minutes after establishing the whole cell
configuration, the rate of recovery slowed down, but the steady-state
current remained the same. Similar effects were seen in five other
Müller cells. This observation can be accounted for by
time-dependent changes in the intracellular milieu of the cell due to
equilibration between the intracellular space and the pipette filling
solution. It was likely that we diluted or washed out some blocking
factor in the cell.
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Probable candidates for intracellular blocking agents are divalent
cations, in particular magnesium ions (Matsuda et al.
1987; Vandenberg 1987
) and polyamines
such as spermine or spermidine (Biedermann et al. 1998
;
Lopatin et al. 1995
). To investigate these
possibilities, we omitted calcium and/or magnesium from the pipette
solution or added ATP to buffer intracellular polyamines (Fakler
et al. 1995
; Watanabe et al. 1991
). Given that
ATP also binds magnesium, we added ATP to a divalent-free solution. In each experiment, the outward current was recorded immediately after
establishing the whole cell recording configuration and 4 min later to
compare the effects of our experimental manipulations to the control
conditions. The results of representative experiments are shown in Fig.
4. When a calcium-free intracellular solution with normal levels of
magnesium was used, the time course of the response did not differ
greatly from that obtained under control conditions (Fig. 4,
A vs. B). However, combined removal of calcium and magnesium produced a decrease in the extent of block in the current
recorded 4 min after establishing the whole cell configuration (Fig.
4C). The decrease in block was further enhanced when ATP was
added to the divalent-free solution as shown in Fig. 4D.
Under these conditions, the increase in the amplitude of the transient was accompanied by a significant slowdown in the rate of decay from the
transient peak to the sustained level.
We next assessed the effects of the different manipulations on the
time-dependent changes in the outward currents (Fig. 4E). When the time constants of the exponential functions fitting the transient currents were calculated for different voltage pulses, we
found that under control conditions the responses were well fit by two
components. A fast component (1) ranging between 2 and 3 ms, and a
second, slower component (
2), ranging between 70 and 180 ms
(n = 5). The data shown in Fig. 4E were
computed from the responses to long 1-s depolarizing pulses in
Müller cells studied after 8 min of whole cell recording with the
divalent-free and 5 mM ATP pipette solution. The time constant of the
fast blocking component increased from about 2 ms to about 10 ms, while
the time constant of the slow blocking mechanism did not change
significantly. When comparing
1 with
2 for the control and ATP
situations, they were significantly different (ANOVA test, at
P < 0.01) when tested at
40, 0, and +40 (mV)
membrane potentials. Likewise, control versus ATP for
1 at these
membrane potential levels were significantly different
(P < 0.01), while
2 control versus ATP were not different.
Differential blocking effects by divalent ions and intracellular
polyamines are reflected in the I-V curves evaluated 25 ms following onset of the voltage pulses. Figure
5A (left) shows the
effects that buffering both magnesium and calcium ions has on the
currents. There is an increase in the slope of the curve for outward
currents with minimal effect on the inward currents. When spermine and
any other polyamines present are buffered by adding ATP to the
divalent-free solution, the outward currents grow even further (Fig.
5A, right). Under these conditions, the relationship between
the outward current and the voltage is nonlinear as indicated in Fig.
5A (right) by the deviation of the I-V
data () from linearity (
). The effects of this solution
are particularly apparent in the physiological range of
60 to +10 mV
and is reduced for more depolarized levels. The inward currents remained relatively unchanged.
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The voltage-dependent changes brought about by divalent-free solutions
with and without ATP are illustrated and quantified by plotting the
corresponding chord conductances (Lopatin et al. 1995)
in Fig. 5B. These data were calculated from the
I-V curves measured 4 min after establishing whole cell
configuration and allowing the intracellular space to equilibrate with
the pipette contents. With a pipette solution with no added calcium
(Calcium-free in Fig. 5B), we observed a depolarizing change
in the blocking voltage, from
74 to
65 mV (SD, 6.4 mV;
P < 0.01, n = 5). With a divalent-free
solution, the corresponding pedestal level rose significantly
(P < 0.01) from 0.43 ± 0.01 (n = 8) to 0.52 ± 0.03 (n = 6) without a major change
in the blocking voltage, which, similar to control values, averaged
72 ± 2.9 mV (n = 6). This last observation is
apparently in conflict with the depolarizing shift of the conductance
observed in the figure, but probably arises as a result of fitting data
of unequal pedestal levels with Boltzmann functions. In the figure, the
conductance obtained under divalent-free conditions does not overlap
the control conductance, but rather its voltage-dependent portion
traces over the conductance obtained under
Ca2+-free conditions, until it diverges due to
the unequal pedestal levels. As a consequence of this "truncation"
of the voltage-dependent portion of the conductance, the Boltzmann
function tends to underestimate the blocking voltage. The overlap of
the Ca2+-free and divalent-free conductances
suggests that omitting intracellular calcium shifts the blocking
potential in the depolarizing direction by approximately 10 mV while
magnesium ions contribute to the pedestal level under these conditions.
We next studied the effects resulting from a reduced concentration of
polyamines. Buffering the intracellular levels of free polyamines with
ATP in a divalent-free solution significantly increased the blocking
voltage to 37 ± 7.0 mV (P < 0.01, n = 5) and the pedestal conductance level to 0.60 ± 0.1 (P < 0.01, n = 5). The value of
the pedestal level does not differ significantly from that obtained
using divalent-free solutions, nor is there a significant change in the
Boltzmann coefficients (1.34 ± 0.16, n = 5) in
ATP solutions. This parallel shift of the conductance is consistent, to
a first approximation, with the decrease in block expected when the
concentration of the blocking agent is reduced (Lopatin et al.
1995
).
In summary, our results indicate that calcium ions had a minor effect on the blocking voltage, magnesium ions had their major impact on the pedestal level (vertical arrow in Fig. 5B), whereas polyamine concentration determined the blocking voltage (horizontal arrow in Fig. 5B). It appears that changes in magnesium ions and polyamines alter the I-V curves and the chord conductances differentially. It is possible that the difference arises from the fact that we may be more effectively buffering the divalent ions than the polyamines. This would shift the blocking voltage to levels beyond +40 mV, and we might then only observe a change in the pedestal conductance. The changes in intracellular concentration of divalent ions or polyamines affected primarily the initial portion of the responses and did not significantly change the I-Vs or chord conductances evaluated at the end of long (1 s) duration pulses (data not shown).
If the reduction in the concentration of free intracellular polyamines
(by buffering with ATP) promotes a decrease in the block of outward
currents and a slow-down in the voltage-dependent block of the
K+ channels, then raising the level of
intracellular polyamines should induce the opposite effects. To test
this prediction, we added spermine to the pipette filling solution
(Bianchi et al. 1996). Only marginal reductions of the
initial transient currents were observed with concentrations under 100 µM. However, with 500 µM spermine in the pipette, significant
changes were observed in the current responses to depolarizing and
hyperpolarizing voltage pulses as shown by representative recordings in
Fig. 6. With spermine added, the initial
transient in the outward current response disappeared, and only
sustained currents remained (Fig. 6A), as is evident by
comparing responses to depolarizing voltage pulses (Fig.
6B). This effect is consistent with a speeding up of the
development of the block.
|
In addition to its effects on outward currents, added spermine also
reduced the magnitude of the inward currents (Fig. 6A). As a
consequence, the regular spacing between the outward currents extends
to the smaller inward currents elicited by voltage pulses to 90 and
100 mV, as rectification is reduced and the currents are reminiscent
of an ohmic conductance. These effects were more clearly demonstrated
by applying hyperpolarizing pulses from a holding potential of
40 mV.
Under these conditions the currents are characterized by an
instantaneous onset followed by a slow relaxation (Fig. 6, C
and D). Raising intracellular spermine affected primarily
the slow phase, which decreased in amplitude in a voltage-dependent manner. For a hyperpolarizing step to
100 mV, the relaxation phase
almost completely disappeared due to the blocking action of spermine
(Fig. 6C), and only a sustained current with amplitude similar to the instantaneous component seen under normal conditions remained. With a step to a more hyperpolarized level (
120 mV), the
block by added spermine is partly overcome, and a small relaxation of
the inward current ensues (Fig. 6D). The I-V
curves measured at 25 ms clearly indicate a reduction of the outward
currents compared with the normal conditions (Fig.
7A). Note that with added
spermine the remaining currents are no longer dependent on holding
potential as the respective I-Vs overlap with each other at
the two different holding potentials.
|
The instantaneous onset of the inward currents is marginally affected
by the added spermine, certainly to a lesser degree than the relaxation
phase (Fig. 6, C and D). Evaluation of the I-V curve 4 ms after onset of the voltage pulses, early
during the relaxation phase, close to the so-called "pseudo
instantaneous phase" (Ishihara et al. 1989), reveals
that rectification is much less marked (Fig. 7B). In fact,
the I-V can be well fit by a single straight line fitting
the outward currents and extending to the inward currents, suggesting
that both inward and outward currents remaining after the block by
spermine may flow through the same, linear channel. In other
experiments, the spermine level was raised to 1 mM and even 2 mM. The
higher concentrations of spermine were not well tolerated by the cells,
but the results were basically similar to those shown in Figs. 6 and 7.
There is the possibility that the instantaneous current arises from the
fast unblock by magnesium ions (Ishihara et al. 1989
).
However, in our experiments, this component remained unaltered when a
divalent-free intracellular solution was used (results not shown).
The corresponding plots of the chord conductances as shown in Fig. 7,
C and D, illustrate the voltage-dependent changes
brought about by exogenous spermine. Addition of 500 µM spermine to
the pipette solution results in a hyperpolarizing shift of the
conductances. Despite a holding membrane potential of 80 mV, the
blocking voltages have shifted significantly to
140 ± 4.2 mV
(P < 0.01, n = 5) at 4 ms,
113 ± 6.9 mV (P < 0.01, n = 5) at 25 ms
(Fig. 7C), and
119 ± 15 mV (P < 0.01, n = 5) for the conductance computed at 1 s
(Fig. 7D). This is indicative of a fascilitatory effect on the block (Lopatin et al. 1995
). The Boltzmann
coefficient has similar values when computed at 25 ms (1.63 ± 0.24, n = 5) and at 1 s (1.86 ± 0.63, n = 5) but is shallower (0.68 ± 0.26, n = 5) when computed at 4 ms. It is not clear whether
this is due to an artifact arising from the shift of the
voltage-sensitive portion of the conductance beyond the range of
voltages applied in these experiments. The pedestal levels measured at
4 ms (0.33 ± 0.04, n = 5), 25 ms (0.33 ± 0.05, n = 5), or 1 s (0.31 ± 0.03, n = 5) are similar in value and overlap (Fig.
7C). These values are not significantly different from the
pedestal value obtained under control conditions in response to long
voltage pulses (1 s; Fig. 7D). This suggests that the
contribution of the added spermine (0.5-2 mM) to the sustained block
of the outward currents is only marginal and primarily speeds up the
rate of block of the transient currents. Its action on the inward
currents is different, affecting primarily the development and
magnitude of the time-dependent component. The compound effects of
spermine on the inward and outward currents translated primarily into a
hyperpolarizing shift of the blocking voltage without a change in the
pedestal level, thus extending the range of linear operation (Fig.
7D), as is implied by the extended range of the pedestal
value. This range is further extended when the conductances are
computed at 4 ms (Fig. 7C), when the conditions for block by
the added spermine are maximized.
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DISCUSSION |
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Turtle Müller cells are highly and almost exclusively
permeable to potassium ions (Conner et al. 1985;
Linn et al. 1998
). On membrane polarization, several
components of the potassium currents are easily discerned. There is a
moderate rectification of inward currents, and for depolarizations an
initial transient, and then a later, sustained component to the outward
currents. These features do not arise as a result of intrinsic
properties of the membrane ion channels but rather reflect the blocking
action of intracellular polyamines and divalent ions.
Time-dependent and ohmic components
The transient outward current that was observed on depolarization
of turtle Müller cells was not due to a voltage-activated current, such as the IA current found
in Müller cells of salamander (Newman 1985) and
rabbit (Nilius and Reichenbach 1988
). In turtle, the
outward current is insensitive to TEA and 4-AP (Linn et al. 1998
) but is strongly dependent on holding potential (Fig. 3), and on the level of intracellular divalent cations and polyamines. Removing calcium ions induced a modest effect on the current (Fig. 4,
A and B); but, when both calcium and magnesium
ions were removed from the intracellular space, the outward currents
greatly increased in magnitude while the inward currents were hardly
affected (Fig. 4C). This increased the slope of the
I-V relationship (Fig. 5A), which translated into
a change in the pedestal levels of the chord conductance. This suggests
a blocking action by magnesium ions on the potassium channels. However,
we did not observe significant changes in the steady-state currents
measured in response to long 1-s pulses (data not shown). The role for
magnesium ions in the short-term block and its diminishing effect with
time is suggestive of a competitive block between magnesium and
polyamines (Ishihara 1997
). Without divalent ions and
with ATP chelating intracellular polyamines, the outward transient
currents increased in amplitude, and their rate of decay slowed down
considerably, particularly affecting the fast component of the block
(Fig. 4). These observations indicate that the transient outward
current may arise from the voltage- and time-dependent block of the
potassium channels by magnesium and polyamines. When expressed in terms
of the chord conductance, these changes in the block of the potassium
currents translated primarily into a shift in the blocking voltages
(Fig. 5B). Lower concentrations of intracellular spermine
require increasingly depolarized voltages to achieve similar degrees of
block. This is similar to findings in expressed HRK1 channels
(Lopatin et al. 1995
), confirming that the
effect of ATP is to lower the concentration of free intracellular
spermine. Note that ATP also binds spermidine, another blocker of
inward rectifier channels (Lopatin et al. 1994
), although with lesser efficacy than spermine (Watanabe et al.
1991
). Given that Müller cells also produce spermidine
(Biedermann et al. 1997
), the effects we describe here
resulting from the buffering action of ATP do not necessarily imply
that we are observing the blocking action of spermine exclusively
(Ishihara et al. 1996
).
When spermine, which was found to be a highly effective blocker of the
Kir channels in rabbit Müller cells
(Biedermann et al. 1998), was added to the
pipette solution at a concentration of 500 µM, the rate at which the
block of the outward current developed was sped up considerably, so
much so that the initial current transient disappeared (Fig. 6), and
the block appeared to be instantaneous. Under these conditions, we
observed very little change in the sustained current levels. Thus under
normal conditions, the intracellular constituents that block outward potassium currents (divalent ions, polyamines) are at a concentration sufficient to produce maximal block; adding spermine only sped up the
development of the block.
Addition of spermine also affected the inward currents. There was
little effect on the instantaneous, onset portion of the current. But
there was a reduction of the time-dependent, slowly developing portion
of the inward current, with consequent reduction of the rectifying
properties (Figs. 6 and 7). The compound effect of the added spermine
on the inward and outward currents produced a parallel shift of the
conductance toward hyperpolarizing voltages, extending the range of the
pedestal level (Fig. 7, C and D), and thereby of
the linear or ohmic conductance to voltages hyperpolarized to 100 mV.
In summary, we can distinguish two kinetically distinct components to
the Müller cell potassium conductance: a time-dependent, rectifying component sensitive to spermine, and an ohmic component that
is marginally sensitive to spermine.
One or two potassium channel types
The question remains as to whether the time-dependent and the
ohmic current components that were observed here flow through the same
channel or through physically distinct potassium channel types
(Solessio et al. 2000). If turtle Müller cells are
similar to those isolated from the guinea pig, then spermine would be expected to abolish outward currents through the inward rectifier channels (Biedermann et al. 1998
) (note, however, that
those experiments were performed with symmetric concentrations of high
potassium, a condition that leads to the enhancement of inward currents
relative to the outward currents, see our Fig. 1). Based on this
assumption, the spermine-sensitive current represents the current
carried through inwardly rectifying potassium channels, blocked by
polyamines by way of a plugging mechanism (Lopatin et al.
1995
), and that are characterized by two apparent binding sites
for block by intracellular barium ions under hyperpolarized conditions
(Solessio et al. 2000
). In such a case, occupancy of the
channels and block by spermine (or other intracellular polyamines)
during depolarization would interfere with binding of the barium ions.
The ohmic current could be carried through a different type of
potassium channel that was blocked by intracellular barium ions
occupying a single site in the channel pore (Solessio et al.
2000
). This current is not sensitive to other known blockers of
potassium channels (TEA and 4-AP) or changes in intracellular calcium,
suggesting that it is not a typical delayed rectifier and not a
calcium-dependent potassium current (Bringmann et al.
1997
). These considerations give credence to the idea that
potassium currents in turtle Müller cells flow through two types
of channels: an inward rectifier and an ohmic conductance.
However, other properties of the currents are not readily reconcilable
with the two-channel model, and we cannot discard the possibility that
all the potassium currents flow through weakly rectifying potassium
channels (Fakler et al. 1994; Krapivinsky et al.
1998
; Kubo et al. 1996
; Takumi et al.
1995
). For example, the ratio of the linear, or ohmic,
component to the rectifying component was the same from cell to cell
and from preparation to preparation, as indicated by the constancy of
the pedestal level. We also found that the same currents were observed
in different parts of the cells, such as the soma and the processes
(Perlman et al. 2001
). This indicates that if the linear
and rectifying components flow through separate channels, then these
channels are linked or coupled as is the case for a tandem pore
potassium channel (Chavez et al. 1999
). Evidence from
Müller cells in rat, rabbit, and mouse (Ishii et al.
1997
; Kofuji et al. 2000
; Tada et al.
1998
) indicates that Müller cells express Kir4.1 inward rectifiers. In fact, the properties of the whole cell currents in
turtle Müller cells bear close resemblance to those recorded from
HEK293T cells expressing Kir4.1 channels (Tada et al.
1998
), suggesting that the potassium currents in turtle
Müller cells flow through a single type of channel, probably
belonging to a subfamily of Kir4.1 channels. This is also compatible
with the observed sensitivity of these currents to block by
extracellular barium (10- to 100-µM range) (Solessio et al.
2000
).
If this is the case, then the sustained currents measured in the
presence of high intracellular spermine levels (Fig. 7) indicate that
the inward rectifier channel of the turtle Müller cells behaves
like they have a wide inner pore, where cations do not flow in single
file (Lu et al. 1999) and that can be completely blocked
by spermine by way of a plugging mechanism (Lopatin et al.
1995
). In this context, the time-dependent components reflect the binding and unbinding of polyamines within the channel pore, whereas the ohmic component represents the residual conductance remaining in the channels after polyamines bind in the pore (Lu et al. 1999
). Assuming that the channel pore can accommodate
more than one blocking particle (Lu et al. 1999
;
Yang et al. 1995
), and/or possess what appears to be two
binding sites, this model can also explain the blocking effects of
barium (Solessio et al. 2000
). That is, on
hyperpolarization, two apparent binding sites are available for barium
ions within the channel pore. With depolarization, only one binding
site is available for occupancy by barium, with the other apparent site
"occupied" by a polyamine molecule that has been driven into the
channel. While this is the most parsimonious explanation of our
results, further studies will be needed to determine whether this is
truly the case.
Potassium siphoning
Regardless of the specific ionic conductances underlying the
I-V relationship of turtle Müller cells, the
properties of the cell's potassium currents suit the role of the
Müller cell in maintaining potassium homeostasis by siphoning
potassium in the turtle retina. Like channels in Müller cells of
salamander (Newman 1993) and in the endfeet of the
Müller cells in frog (Skatchkov et al. 1995
), the
channels in turtle Müller cells provide the route for potassium
flux in both directions. During light-induced neuronal activity,
potassium can flow into the cells in regions where extracellular
potassium increases and out of the cell in other areas, particularly
the endfeet.
Our work suggests that intracellular polyamines provide the
Müller cell with a biochemical mechanism with which to regulate the properties of their potassium channels. It is intriguing that varying the intracellular concentration of polyamines differentially affected the various components of the potassium currents. Alterations in polyamine (spermine) concentration predominantly affected the time
course of the currents elicited at the onset of the depolarization (changing the time constant of block) and the time-dependent component of the inward currents. We had to use a concentration of spermine of
0.1-0.5 mM in the pipette to reliably block the currents. This is a
relatively low concentration, considering the reported concentrations of intrinsic spermine of about 0.25 mM in RBL-1 cells (Bianchi et al. 1996), 1.57 mM in bovine lymphocytes (Watanabe et
al. 1991
), and 0.1-0.3 mM in Xenopus oocytes
(Lopatin et al. 1994
). Values of spermine in
Müller cells have not yet been reported. But it is interesting
that at resting levels, the free concentration of polyamines
(Watanabe et al. 1991
) seems to reach an equilibrium whereby it is sufficient to produce a maximal block of the outward currents (with prolonged depolarizations) without altering the magnitude of the sustained inward currents. Had the levels been higher,
the time-dependent components would be reduced and the rectifying
properties of the currents compromised.
It is probably of some importance for normal retinal physiology that
spermine can differentially modulate various components of the
Müller cell potassium currents and, as a result, change the
relative contribution of each to siphoning. Polyamines are synthesized
from arginine or ornithine along a highly regulated pathway and are
transported into and out of the cell as well. One can imagine metabolic
states (light or dark adaptation), where synthesis and/or transport are
up and down regulated (Morgan 1999). Such changes on a
relatively rapid time scale have been described in the mouse retina
(Macaione et al. 1993
). This regulation in turn would
facilitate a decrease or increase in potassium influx, respectively. If
this is closely coupled to increases or decreases in extracellular
potassium levels, then a mechanism exists for fine tuning the process
of siphoning.
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ACKNOWLEDGMENTS |
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This work was supported by National Eye Institute Grant EY-05972 to E. M. Lasater and by an unrestricted grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology and Visual Sciences, University of Utah.
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FOOTNOTES |
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Address for reprint requests: E. M. Lasater, John Moran Eye Center, Dept. of Ophthalmology and Visual Sciences, 50 North Medical Dr., Salt Lake City, UT 84132 (E-mail: eric.lasater{at}hsc.utah.edu).
Received 16 March 2000; accepted in final form 18 December 2000.
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REFERENCES |
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