From the Department of Physiology and Graduate Program in Biophysics, School of Medicine, University of California at San Francisco, San Francisco, California 94143
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ABSTRACT |
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The selectivity of Ca2+ over Na+ is ~3.3-fold larger in cGMP-gated channels of cone photoreceptors
than in those of rods when measured under saturating cGMP concentrations, where the probability of channel
opening is 85-90%. Under physiological conditions, however, the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination.
We investigated the ion selectivity of cGMP-gated channels as a function of cyclic nucleotide concentration in
membrane patches detached from the outer segments of rod and cone photoreceptors and have found that ion
selectivity is linked to gating. We determined ion selectivity relative to Na+ (PX/PNa) from the value of reversal
potentials measured under ion concentration gradients. The selectivity for Ca2+ over Na+ increases continuously
as the probability of channel opening rises. The dependence of PCa/PNa on cGMP concentration, in both rods
and cones, is well described by the same Hill function that describes the cGMP dependence of current amplitude.
At the cytoplasmic cGMP concentrations expected in dark-adapted intact photoreceptors, PCa/PNa in cone channels is ~7.4-fold greater than that in rods. The linkage between selectivity and gating is specific for divalent cations. The selectivity of Ca2+ and Sr2+ changes with cGMP concentration, but the selectivity of inorganic monovalent cations, Cs+ and NH4+, and organic cations, methylammonium+ and dimethylammonium+, is invariant with
cGMP. Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies of and
subunits. The maximal PCa/PNa of channels formed from
subunits of bovine rod channels is less than that of heteromeric channels formed from
and
subunits. In addition, Ca2+ is a more effective blocker of channels formed by
subunits than of channels formed by
and
subunits. The cGMP-dependent shift in divalent cation selectivity
is a property of
channels and not of channels formed from
subunits alone.
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INTRODUCTION |
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The essential functions of ion-conducting channels, selectivity and gating, have historically been described as
independent processes, each determined by different
features of the molecular structure of the channels
(thoroughly reviewed by Andersen and Koeppe, 1992).
Direct investigation of cloned channels, however, has
demonstrated that these functions may be linked. In
voltage-gated K+ channels, for example, the mutation
of a single amino acid can change both the ion selectivity and the voltage dependence of activation (Yool and
Schwarz, 1991
; Heginbotham and MacKinnon, 1992
).
In N-methyl-D-aspartate channels, ion selectivity changes with the state of channel activity (Schneggenburger
and Ascher, 1997
); this is also the case in some voltage-gated K+ channels (Zheng and Sigworth, 1997
). Because of structural similarities, cGMP-gated ion channels are recognized as members of the superfamily of
voltage-gated ion channels (reviewed in Zagotta and
Siegelbaum, 1996
). In cGMP-gated channels, there is
no direct evidence that ion selectivity is linked to the
state of channel activity. However, Cervetto et al. (1988)
have reported that the relative ability of various divalent cations to carry current through these channels in
intact rod photoreceptors appears to change as a function of cGMP concentration.
The activity of cGMP-gated channels underlies the
light-dependent conductance of rod and cone photoreceptors in the vertebrate retina. These channels select
divalent over monovalent cations (rods: Colamartino
et al., 1991; Zimmerman and Baylor, 1992
; Wells and
Tanaka, 1997
; cones: Picones and Korenbrot, 1995
;
Haynes, 1995
), although they select poorly among
monovalent cations (rods: Furman and Tanaka, 1990
; Menini, 1990
; cones: Picones and Korenbrot, 1992
; Haynes,
1995
). The relative selectivity of Ca2+ over Na+ (PCa/
PNa) is higher in cone than in rod channels, both in
native membranes (Haynes, 1995
; Picones and Korenbrot, 1995
) and in recombinant channels formed from
subunits alone (Frings et al., 1995
). This difference
may be important in understanding the difference in phototransduction signals between the two receptor
types, because the Ca2+ influx through these channels,
and its balance with efflux via a Na+/Ca2+,K+ exchanger, helps maintain the cytoplasmic free Ca2+ concentration in these cells (Yau and Nakatani, 1985
;
Miller and Korenbrot, 1987
). The differences in PCa/
PNa between cones and rods make it likely that light-dependent changes in cytoplasmic Ca2+ caused by the
same light intensity will be larger and faster in cones
than in rods (Miller and Korenbrot, 1994
; Korenbrot, 1995
). Studies of ion selectivity in cGMP-gated channels of photoreceptor membranes, however, have only
been conducted at ligand concentrations that fully activate the channels. Yet, in intact photoreceptors and under physiological conditions, at most 1-5% of the
cGMP-gated channels are open (Cobbs et al., 1985
;
Hestrin and Korenbrot, 1987
; Cameron and Pugh,
1990
), which indicates that the highest cytoplasmic
concentration of cGMP in the cells is about four- to
five-fold smaller than K1/2, the nucleotide concentration that half-saturates current amplitude. If ion selectivity were linked to gating, then the channel attributes
in the intact photoreceptor might differ from those
known from studies under saturating agonist concentrations.
We investigated the ion selectivity of cGMP-gated
channels from both rods and cones as a function of
cGMP concentration in membrane patches detached
from intact outer segments. Contrary to the traditional
view, we have found that the selectivity is indeed linked
to gating: the selectivity for Ca2+ over Na+ increases
continuously as the probability of channel opening rises. This proportionality is steeper in channels of rods
than in those of cones. Under physiological cGMP concentrations, PCa/PNa in cone channels is ~7.5-fold
larger than that in rod channels, significantly larger
than had been previously measured at saturating concentrations of cGMP (Picones and Korenbrot, 1995).
Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies composed of at least
two structural subunits, and
(Chen et al., 1993
; Korschen et al., 1995
). By homology, it is likely that channels in cones also comprise
and
subunits, but such
subunits have not been identified to date.
subunits of
bovine rod and cone channels expressed in Xenopus oocytes preserve the differences in Ca2+ selectivity characteristic of native channels, but the ability of Ca2+ to
block the channel and the absolute values of PCa/PNa
differ from those of native channels (Frings et al., 1995
;
Picones and Korenbrot, 1995
). This suggests that the
selectivity and interaction of Ca2+ with cGMP-gated
channels may depend on the interaction of
and
subunits. Using Xenopus oocytes as an expression system, we determined that the cGMP-dependent shift in
divalent cation selectivity is a property of heteromeric
channels and not of homomeric channels formed
from
subunits alone.
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MATERIALS AND METHODS |
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Materials
Striped bass (Morone saxitilis) were obtained from Professional Aquaculture Services and maintained in the laboratory for up to 6 wk under 10:14-h dark:light cycles. Tiger salamanders (Ambystoma tigrinum) were received from Charles Sullivan and maintained in the laboratory in an aquarium at 6°C under 12:12-h dark:light cycles. The UCSF Committee on Animal Research approved protocols for the upkeep and killing of the animals. l-cis-diltiazem was the kind gift of Tanabe Seiyako Co. Ltd.
Photoreceptor Isolation
Under infrared illumination and with the aid of a TV camera and
monitor, retinas were isolated from dark-adapted animals and photoreceptors were dissociated as described in detail elsewhere (Miller and Korenbrot, 1993, 1994
). Single cones were isolated by mechanical dissociation of fish retinas briefly treated with collagenase and hyaluronidase. Solitary cones were maintained in a
Ringer's solution consisting of (mM): 143 NaCl, 2.5 KCl, 5 NaHCO3, 1 Na2HPO4, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES,
pH 7.5, osmotic pressure 309 mOsM. Rod outer segments were
isolated by mechanical dissociation of tiger salamander retinas
and were maintained in a Ringer's solution composed of (mM):
100 NaCl, 2 KCl, 5 NaHCO3, 1 Na2HPO4, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH 7.5, osmotic pressure 227 mOsM.
Solitary photoreceptors were firmly attached to a glass coverslip derivatized with wheat germ agglutinin (Picones and Korenbrot, 1992). The coverslip formed the bottom of a recording
chamber held on the fixed stage of an upright microscope
equipped with DIC optics and operated under visible light. A suspension of photoreceptors in Ringer's in which glucose was replaced with 5 mM pyruvate was added to the recording chamber
and cells were allowed to settle and attach to the coverslip. After
5 min, the bath solution was exchanged with the normal, glucose-containing Ringer's.
The recording chamber consisted of two side-by-side compartments. Cells were held in one compartment that was continuously perfused with glucose containing Ringer's. The second,
smaller compartment was continuous with the first one, but a
movable barrier could be used to separate them (Picones and
Korenbrot, 1992). We used tight-seal electrodes to obtain inside-out membrane fragments detached from the side of the outer
segments of either cones or rods. Electrodes were produced
from aluminosilicate glass (1.5 × 1.0 mm o.d. × i.d., 1724; Corning Glass Works). After forming a giga-seal and detaching the
membrane fragment, the electrode was moved under the solution surface from the compartment containing the cells to the
smaller compartment. The barrier was moved to isolate the two
compartments and the electrode tip was placed within 100 µm
from the opening of a 300-µm diameter glass capillary that delivered test solutions onto the cytoplasmic (outside) surface of the
membrane patch. In a significant fraction of patches, we initially
failed to observe cGMP-activated currents. We assumed these
were closed vesicles since we frequently succeeded in eliciting currents after rapidly crossing the air-water interface.
Expression of Channels in Xenopus Oocytes
Plasmids containing either the or
subunits of the bovine rod
cGMP-gated channel flanked by 5' and 3' untranslated regions of
the Xenopus
-globin gene (Liman et al., 1992
) were kindly provided by the laboratories of W. Zagotta (University of Washington, Seattle, WA) and R. Molday (University of British Columbia,
Vancouver, British Columbia, Canada), respectively. Capped
RNA was transcribed from the linearized plasmid with T7 RNA
polymerase (Swanson and Folander, 1992
). RNA was purified by
extractions with phenol/chloroform, recovered by ethanol precipitation and dissolved in RNAase-free water at a concentration
of 2 µg/µl. Xenopus laevis oocytes, generously provided by the lab
of L.Y. Jan (University of California at San Francisco) were each
injected with ~45 nl of RNA (90 ng). When
and
subunits
were coinjected, RNA was mixed at a weight ratio of 4:1 (
:
). Injected oocytes were gently rocked at 18°C in ND96 media supplemented with 2.5 mM sodium pyruvate, 100 U/ml penicillin, and
100 µg/ml streptomycin. Oocytes were suitable for electrical studies 3-5 d after injection. Immediately before patch clamping, each oocyte was incubated for 5 min in a hypertonic solution
composed of (mM): 200 NaCl, 10 HEPES, 1 CaCl2, 1 MgCl2, pH
7.5, and its vitelline membrane removed. Denuded oocytes were
attached to clean glass coverslips in the recording chamber described above and bathed in ND96. We used tight-seal electrodes
to obtain inside-out detached membrane patches. The electrodes
were produced from aluminosilicate glass (1.5 × 1.0 mm o.d. × i.d.) with large tip openings (~2 µm).
Ionic Solutions
In studies of both rod and cone photoreceptor membranes, we
filled the tight-seal electrodes with the same, standard solution (mM): 150 NaCl, 5 BAPTA, 10 HEPES, adjusted with tetramethylammonium hydroxide (TMA-OH) to pH 7.5, osmotic pressure
300 mOsM. TMA-OH was used to titrate pH in all solutions because TMA does not permeate the channels of rods or cones (Picones and Korenbrot, 1992; Picco and Menini, 1993
). Free Ca2+
concentration in this solution was <10
10 M. After detachment,
all membrane patches were first exposed for at least 2 min to a
standard solution composed of (mM): 150 NaCl, 1 EDTA, 1 EGTA, and 10 HEPES, adjusted with TMA-OH to pH 7.5. This solution thoroughly removed any endogenous modulator that
might remain associated with the channels (Hackos and Korenbrot, 1997
). Ionic concentrations were calibrated by measuring
the osmotic pressure of the solutions and comparing it with published standards (Weast, 1987
).
The solution bathing the cytoplasmic membrane surface was
selected among four possible test conditions, depending on the
objective of the experiment: (a) the standard solution defined
above; (b) the standard solution containing varying concentrations of cGMP; (c) the standard solution containing varying concentrations of Ca2+ or other divalent cations, with or without
cGMP; and (d) solutions containing 150 mM of various monovalent cations replacing Na+ in the standard solution, with or without cGMP. We began every experiment by measuring current-
voltage (I-V)1 curves under symmetric NaCl solutions first in the
absence, and then in the presence, of 1 mM cGMP. The point at
which these two curves intersect defined the origin (0,0) in the
I-V plane for all measurements in that membrane patch. At the
end of experimental manipulations, these curves were again
measured. We only analyzed data from membrane patches in
which the origin did not shift and in which the maximum cGMP-dependent conductance changed by 10%.
Internal and external solutions used to study membrane
patches detached from Xenopus oocytes were similar to those
used in studies of photoreceptor membranes, except that Cl was
replaced with methanesulfonate to eliminate the Ca2+-activated
Cl
current characteristic of the oocyte membranes (Miledi and
Parker, 1984
). To use a Ag/AgCl electrode in the absence of Cl
ions, we modified the tight-seal electrode holder to incorporate a
1-M KCl/agar bridge between the electrode-filling solution and the Ag/AgCl half-cell.
Electrical Recordings
We measured membrane currents under voltage clamp at room
temperature with a patch clamp amplifier (8900; Dagan Corp.).
Analogue signals were low-pass filtered below 1 kHz with an
eight-pole Bessel filter (Frequency Devices Inc.) and were digitized on line at 3 kHz (FastLab; Indec). Membrane voltage was
normally held at 0 mV and membrane currents were activated
with continuous voltage ramps from 70 to +70 mV (over 1 s) or
from
30 to +30 mV (over 0.5 s). In experiments with biionic solutions of monovalent cations, the holding voltage was set at the
reversal potential corresponding to the ionic condition under investigation. We did so to avoid current flow at the holding voltage
in order to minimize errors due to ion accumulation (or depletion) within the electrode's tip. Before initiating the voltage
ramp, the voltage was held for 200 ms at either
70 or
30 mV
(depending on the ramp range). This interval was sufficient to
attain a steady current at
70 or
30 mV, following the time-
dependent changes due to the relief by voltage of divalent cation
channel block (rods: Colamartino et al., 1991
; Zimmerman and
Baylor, 1992
; cones: Picones and Korenbrot, 1995
). A Ag/AgCl
reference electrode was connected to the bath through a 1-M
KCl agar bridge to avoid shifts in electrode potential as solutions
changed. As is conventional, outward currents are positive and
the extracellular membrane surface was defined as ground.
Permeability Calculations
We calculated permeability ratios from reversal potentials using
the Goldman-Hodgkin-Katz constant field equation. To determine reversal potentials of cGMP-activated currents, a straight
line was fit to their I-V curve between 15 and +10 mV. Reversal
was that potential at which this straight line intercepted the I-V curve measured in the same patch and under the same ionic gradient, but in the absence of cGMP (the leak current). Under biionic monovalent cation solutions, ion selectivity was expressed
as a permeability ratio defined by the equation:
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(1) |
where PX/PNa is the permeability ratio of the cation X relative
to Na+, Vrev is the measured reversal potential, [Na]o is the extracellular Na+ activity, and [X]i is the intracellular activity of cation
X. F, R, and T have their usual thermodynamic meanings. Activity coefficients were taken from Robinson and Stokes (1959).
Under conditions of symmetric Na+ with Ca2+ (or other divalent cation) added only to the intracellular side of the membrane, we used the following equation derived from a more general equation given by Lewis (1979):
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(2) |
where [Na] is the Na+ activity on both sides of the membrane,
and [Ca]i is the intracellular Ca2+ activity. Activity coefficients for
Ca2+ in the presence of monovalent cations were taken from Butler (1968), and then squared in accordance with the Guggenheim convention (Robinson and Stokes, 1959
). Activity coefficients for Sr2+ were assumed to be the same as for Ca2+.
Mathematical functions were fit to experimental data using nonlinear, least square minimization algorithms (Origin; Microcal Software, Inc.). Statistical errors are presented throughout as mean ± SD.
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RESULTS |
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The Ca2+ to Na+ Selectivity Ratio in Channels of cGMP-gated Channels of Rod Photoreceptors Depends on Ligand Concentration
We examined cGMP-dependent currents in inside-out
membrane patches detached from the outer segment
of rods isolated from the tiger salamander retina. In
the presence of 300 µM cGMP, a concentration at
which the probability of channel opening is at its maximum value, the currents under symmetric Na+ solutions reversed direction at ~0 mV and their I-V curves
were nearly linear (Fig. 1). Addition of Ca2+ to the cytoplasmic membrane surface shifted the reversal potential to a more negative value and changed the shape of
the I-V curve (Fig. 1), as has been previously reported
(Colamartino et al., 1991; Zimmerman and Baylor,
1992
; Tanaka and Furman, 1993
; Picones and Korenbrot, 1995
). The shift in reversal potential reveals that
the channels are more permeable to Ca2+ than to Na+
and the change in the I-V curve reflects a voltage-dependent block of the channels by Ca2+. The average
shift in reversal potential with 10 mM Ca2+ was
6.3 ± 0.27 mV (range
8.5 to
5.1, n = 7). This indicates (Eq. 2, using ion activities and assuming the Guggenheim convention, see MATERIALS AND METHODS) that
the average value of PCa/PNa is 6.48 ± 0.35.
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We investigated whether the value of PCa/PNa
changed with cGMP concentration. Under symmetric
Na+ solutions with 10 mM Ca2+ added to the cytoplasmic membrane surface, we measured membrane currents generated by voltage ramps in the presence of
various cGMP concentrations (Fig. 2). As has been repeatedly shown before (reviewed in Yau and Chen,
1995; Zagotta and Siegelbaum, 1996
), the amplitude
of the current, at a fixed voltage, increased with cGMP
in a manner well described by the Hill equation (Fig.
2 B).
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(3) |
|
where I is the amplitude of the cGMP-dependent membrane current, Imax is its maximum value, [cGMP] is the concentration of cGMP, K1/2 is that concentration necessary to reach one half the Imax value, and n is a parameter that reflects the cooperative interaction of cGMP molecules in activating the membrane current. Remarkably, the reversal potential of the I-V curves shifted as the cGMP concentration changed (Fig. 2), suggesting that PCa/PNa is not constant, but changes as a function of channel gating.
For experimental convenience and to reduce uncertainties due to potential changes in the leakage of the
tight electrode seal, we used an alternative method to
rapidly change cGMP concentration, which we will refer to as a cGMP concentration ramp. In this protocol, the patch was continuously superfused. In the presence
of symmetric Na+ with 10 mM Ca2+ on the cytoplasmic
membrane surface, we first measured I-V curves activated by fixed concentrations of cGMP up to 300 µM
(Fig. 2). We used these data to generate a current
amplitude-concentration function at a fixed voltage,
+15 mV, for that patch (Fig. 2 B). Next, we imposed a
step change in cGMP concentration to 300 µM and
waited until currents reached a maximum, stationary
value (typically 30 s). We then switched to a solution
free of cGMP and continued superfusing, while repeatedly measuring I-V curves with rapid voltage ramps
(30 to +30 mV/0.5 s) delivered at 2-s intervals. As the
cGMP diffused away from the patch, currents decreased in amplitude (Fig. 2). As expected from simple
diffusion between two compartments, the time course
of cGMP loss was well described by a single exponential
(Fig. 2). We limited our analysis to patches in which the
time constant of this exponential was
12 s. Under
these conditions, we assume that individual I-V curves (each measured in 500 ms) are measured at constant
cGMP. We measured current amplitude at +15 mV in
each ramp I-V curve and, using the calibration data
first generated in the same patch, we established the
cGMP concentration at which each ramp I-V curve was measured.
Using cGMP concentration ramps, we confirmed that the reversal potential measured under a Ca2+ concentration gradient changes with cGMP concentration. The reversal potential shifted progressively towards less negative values as cGMP concentration decreased, revealing that the channels become less selective for Ca2+ over Na+ as their probability of opening decreases (Fig. 3). The dependence of reversal potential, and therefore PCa/PNa (Eq. 2), on [cGMP] was well described by a Hill function modified in the following form (Fig. 3):
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(4) |
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are the asymptotic minimum and maximum values attained by PCa/PNa, K1/2 is the concentration at which
PCa/PNa has a value midway between its maximum
and minimum values, and n is an adjustable parameter
that reflects ligand cooperativity. The average value of
the parameters that best fit our data were K1/2 = 17.2 ± 8.6 µM, n = 2.43 ± 0.37, PCa/PNamax = 6.48 ± 0.35, and PCa/PNamin = 1.89 ± 0.52 (n = 14). In every
patch tested (n = 14), the values of K1/2 and n that best
described the cGMP dependence of PCa/PNa were exactly the same as those that best described the dependence of membrane conductance on nucleotide concentration (Eq. 3). This is illustrated in Fig. 3, where
the change in conductance is plotted as the value of
(1 I/Imax) at +15 mV, where I is the current at a
given cGMP concentration and Imax is its maximum
value. The plot is scaled to have its minimum and maximum values, 0 and 1, respectively, match the minimum
and maximum values of PCa/PNa. While plotting conductance this way is unconventional, since the function
decreases as cGMP concentration increases, it allows us
to compare directly the cGMP dependence of conductance and PCa/PNa. Membrane conductance is a direct measure of the average probability of channel
opening (Picones and Korenbrot, 1994
). In rods, therefore, PCa/PNa changes 3.42 ± 0.95-fold (n = 14) between its minimum and maximum values in a manner
that is directly proportional to the probability of channel activation.
cGMP-dependent Shifts in Reversal Potential Do Not Occur under Symmetric Na+ Solutions
Patches excised from tiger salamander rod outer segments often exhibit cGMP-activated currents as large as
1-2 nA at 70 mV under symmetric Na+ solutions.
These large currents, when sustained over a long time period, display a slow exponential decline in amplitude
caused by the accumulation of Na+ ions at the electrode tip and a consequent, slow shift in reversal potential (Zimmerman et al., 1988
). It was important, therefore, to ascertain that the rapid changes in reversal potential we observed in the presence of symmetric Na+
and a Ca2+ gradient did not arise from Na+ accumulation at the electrode tip. Using cGMP concentration
ramps, we measured the reversal potential under symmetric Na+ solutions as a function of cGMP (Fig. 4). If
Na+ accumulation occurred to a significant extent under our experimental protocol, then the reversal potential should shift in time. In fact, we found that the
reversal potential was time invariant and independent
of [cGMP]. The same results were obtained in every patch we studied with this protocol (n = 21). In solutions containing 10 mM Ca2+, Na+ accumulation
should occur to an even lesser extent since ionic currents are much smaller due to Ca2+-dependent block of
the pore. Furthermore, accumulation of Ca2+ ions on
the extracellular side of the patch cannot occur due to
the presence of 5 mM BAPTA in the patch pipette solution. Thus, the cGMP-dependent shift in reversal potential under Ca2+ concentration gradients reflects
changes in PCa/PNa, and not the generation of an
asymmetry in Na+ or Ca2+ concentrations.
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Effectiveness of Ca2+ Block Is also a Function of cGMP Concentration
Our results reveal the complex interactions between
gating and ion selectivity for Ca2+ ions. Previous reports have documented that the blocking effect of Ca2+
on the channels is also a function of cGMP (Colamartino et al., 1991; Karpen et al., 1993
; Tanaka and Furman, 1993
). Fig. 5 illustrates this phenomenon in our experiments. The extent of conductance block by 5 mM
Ca2+ in the voltage range between
80 and +80 mV was
measured in the same patch at various cGMP concentrations (5, 10, 20, 40, 300 µM). The I-V curves under symmetric Na+ solutions (150 mM) were first measured in
the presence of the various cGMP concentrations using
a voltage ramp. The same curves were then measured
again at each of the cGMP concentrations, but now with
5 mM Ca2+ added to the cytoplasmic surface. The extent
of Ca2+-dependent conductance block, gCa(v)/g(v) was
determined by dividing, for each cGMP concentration tested, the current ramp measured in the presence of
5 mM cytoplasmic Ca2+ by the current ramp measured
in its absence (Fig. 5). At all cGMP concentrations, the
conductance block was maximal near the reversal potential, and it was relieved by either depolarization or hyperpolarization. At any given voltage, the extent of block was a function of cGMP, and this dependence was well
described by the same Hill function that describes the
cGMP dependence of current amplitude (Fig. 5). We obtained the same results in every patch we tested (n = 4).
Thus, changing the probability of channel opening affects not only the ion selectivity of the channel for Ca2+,
but also the interaction between Ca2+ and the pore.
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The Selectivity for Other Divalent Cations over Na+ Is a Function of cGMP Concentration
We explored whether the effect of cGMP on Ca2+ selectivity was specific for this ion or was a feature common
to other divalent cations. We elected to study the selectivity properties of Sr2+, rather than Mg2+, because this
cation permeates the channels, but is a less effective channel blocker than Ca2+ or Mg2+. Using cGMP concentration ramps, we measured the reversal potential under symmetric Na+ solutions with 20 mM Sr2+ added
to the cytoplasmic membrane surface of the patch (Fig. 6). In the presence of saturating cGMP (300 µM), the
reversal potential was 5.3 ± 0.21 mV (n = 8), which
indicates that PSr/PNa = 2.71 ± 0.13. As with Ca2+, the
reversal potential shifted to less negative values as
cGMP decreased (Fig. 6). We calculated PSr/PNa from
the reversal voltage (Eq. 2) and found that the dependence on cGMP of this selectivity ratio was well described by the modified Hill equation (Eq. 4; Fig. 6).
On average, the values of the parameters that best fit our data were: K1/2 = 14.8 ± 6.2 µM, n = 2.6 ± 0.32, PSr/PNamin = 0.64 ± 0.27, and PSr/PNamax = 2.71 ± 0.13 (n = 8). Again, in each instance, the values of K1/2
and n were the same as those that best described the
dependence of current amplitude on cGMP in the
same patch (Eq. 3). Thus, the effects of channel gating on selectivity are not specific for Ca2+.
|
Inorganic Monovalent Cation Selectivity Does Not Change as a Function of cGMP Concentration
While the selectivity among monovalent cations is poor
in the rod channel, there are, nonetheless systematic
differences in selectivity among these ions (Furman
and Tanaka, 1990; Menini, 1990
). We explored
whether channel gating affected the selectivity among
inorganic monovalent cations. We elected to test the effects of cGMP on the selectivity between Cs+ and Na+
or NH4+ and Na+. Cs+ is less permeable than Na+,
while NH4+ is more permeable (Furman and Tanaka,
1990
; Menini, 1990
). Using cGMP concentration
ramps, we measured the reversal potential under biionic conditions of either 150 mM Cs+/150 mM Na+ or
150 mM NH4+/150 mM Na+ (Fig. 7). At 300 µM cGMP,
the reversal potential was 18.1 ± 1.6 mV (n = 8) for
Cs+ and
22.2 ± 2.2 mV for NH4+ (n = 6). These results reproduce those previously reported by others
(Furman and Tanaka, 1990
; Menini, 1990
). From Eq. 1,
we find that PCs/PNa = 0.50 ± 0.03 and PNH4/PNa = 2.44 ± 0.21. Unlike our findings with divalent cations,
the reversal potential under monovalent biionic conditions was constant and independent of cGMP (Fig. 7).
Thus, the effect of gating on ion selectivity is exclusive
for divalent cations.
|
The Organic Monovalent Cation Selectivity Does Not Change as a Function of cGMP Concentration
The relative selectivity of organic cations through the
cGMP-gated channels has been used to asses the steric
hindrance imposed on ion flux by the selectivity filter
in the channel (rods: Picco and Menini, 1993; cones:
Stotz and Haynes, 1996
). To determine whether gating
affects steric hindrance, we investigated the effects of
cGMP on the selectivity between methylammonium
(MA+) and Na+ or dimethylammonium (DMA+) and
Na+. Using cGMP concentration ramps, we measured
the reversal potential under biionic conditions of either 150 mM MA+/150 mM Na+ or 150 mM DMA+/
150 mM Na+ (Fig. 8). At 500 µM cGMP, the reversal potential was 16.5 ± 1.5 mV (n = 4) for MA+ and 52.0 ± 2.2 mV (n = 5) for DMA+ (Fig. 8). Eq. 1 yields relative
selectivities of PMA/PNa = 0.53 ± 0.03 and PDMA/
PNa = 0.13 ± 0.011, in agreement with previous measurements (Picco and Menini, 1993
). As in the case of
inorganic cations, we found no changes in the channel
selectivity for organic monovalent cations as a function
of cGMP (Fig. 8). Thus the apparent pore radius of the
selectivity filter does not appear to change as a function
of channel gating.
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The Effects of Gating on the Ion Selectivity of Recombinant Channels from Rods
Cyclic nucleotide-gated channels in rod photoreceptors and olfactory neurons are heteromeric assemblies
of at least two structural subunits, and
(Chen et al.,
1993
; Bradley et al., 1994
; Liman and Buck, 1994
; Korschen et al., 1995
). We investigated whether the cGMP
dependence of ion selectivity of the native channel is a
feature of the
subunits or requires the coexpression
of
and
subunits. We expressed bovine rod
or
channels in Xenopus oocytes and measured cGMP-
dependent currents in inside-out, detached membrane
patches. As discussed in MATERIALS AND METHODS, we
took precautions to eliminate contamination of the recorded currents by Ca2+-dependent Cl
currents native
to the oocyte. In experiments with
channels, we verified that the channels were indeed formed from both
and
subunits by testing the action of l-cis-diltiazem
(10 µM), which effectively blocks only
heteromeric
channels, and not
homomeric channels (Chen et al.,
1993
; Korschen et al., 1995
) (Fig. 9). Also, our data
confirms that homomeric
channels are more sensitive to block by Ca2+ than
channels (Fig. 9) (Korschen et al., 1995
).
|
We measured I-V curves of cGMP-dependent currents in the presence of symmetric Na+ with 10 mM
Ca2+ added to the cytoplasmic surface of the membrane containing either or
channels (Fig. 10). At
saturating cGMP, the reversal potential for
channels
was
4.8 ± 1.2 mV (n = 7), which implies that PCa/ PNa = 4.6 ± 0.79, but for
channels the reversal potential was
8.4 ± 1.1 mV (n = 12), which implies that
PCa/PNa = 9.4 ± 1.1. Thus the heteromeric
channel is about twice more selective for Ca2+ over Na+ than
the
homomeric channel.
|
More remarkable, however, is the difference between
the channels on the effect of gating on ion selectivity.
In channels, PCa/PNa was invariant with cGMP concentration in every patch we tested (n = 8) (Fig. 10). In
channels, as in native channels, the divalent cation
selectivity decreased as cGMP concentration was lowered (Fig. 10). The dependence of PCa/PNa on cGMP
was well described by the modified Hill equation, (Eq. 4). The average value of the parameters in the equation
that best fit our data were: K1/2 = 45.2 ± 15.6 µM, n = 2.6 ± 0.24, PCa/PNamax = 9.4 ± 1.1, PCa/PNamin = 4.4 ± 1.4 (n = 12).
To determine whether the shifts in selectivity for the
channel were specific for divalent cations, we tested
the selectivity for Cs+ and NH4+ in both
and
channels. In Fig. 11, we illustrate I-V curves of cGMP-dependent currents measured in the presence of biionic solutions of Cs+/Na+ or NH4+/Na+. The I-V curves were
generally similar to those recorded from native rod
channels under comparable conditions (see Fig. 7), except that
channels in the presence of biionic solutions
of Cs+/Na+ display an unusual nonlinearity at potentials
more negative than the reversal potential. In the presence of saturating cGMP, the selectivity sequence of
was the same as
, although the absolute values of PX/
PNa differed between the two channels. Mean values of
Vrev for the
channel were Cs+, 28.6 ± 2.5 mV (n = 8);
NH4+,
31.2 ± 1.8 mV (n = 8), which yields PCs/PNa = 0.33 ± 0.03 and PNH4/PNa = 3.49 ± 0.25. Mean values
of Vrev for the
channel were Cs+, 21.9 ± 1.9 mV (n = 6); NH4+,
24.5 ± 2.2 mV (n = 6), which indicate that
PCs/PNa = 0.43 ± 0.32 and PNH4/PNa = 2.67 ± 0.23. The reversal potentials were the same at all cGMP concentrations tested (Fig. 11). Thus, recombinant
or
channels, just like native channels, select poorly among monovalent cations, and this selectivity is unaffected by
gating. However, the absolute value of the selectivity
among monovalent cations, just as among divalents, differs in homomeric and heteromeric channels.
|
Relative Ca2+ Permeability also Depends on cGMP in Channels of Cone Photoreceptors
cGMP-gated channels of cone photoreceptors are significantly more permeable to Ca2+ than those of rods
(Frings et al., 1995; Haynes, 1995
; Picones and Korenbrot, 1995
). However, past measurements were conducted under saturating cGMP concentrations. If channels in cones, like those of rods, change divalent cation
selectivity as a function of cGMP, then differences observed under high cGMP concentrations might not occur under lower concentrations, such as those expected in the photoreceptor cells under physiological
conditions. We investigated the effects of cGMP on the
value of PCa/PNa in cGMP-gated channels from the
striped bass single cone. Using cGMP concentration ramps, we measured the reversal potential under symmetric Na+ solutions with 5 mM Ca2+ added to the cytoplasmic membrane surface (Fig. 12). At saturating cGMP, the reversal potential was
9.4 ± 0.5 mV (n = 12), which implies that, in cones, PCa/PNa = 21.7 ± 1.65 (n = 12), consistent with earlier measurements
(Picones and Korenbrot, 1995
). As in rods, the reversal
potential shifted to less negative values as cGMP concentration declined (Fig. 3). Again, the dependence on cGMP of PCa/PNa was well described by the modified
Hill equation, (Eq. 4; Fig. 12). On average, we found that
K1/2 = 43 ± 14 µM, n = 2.8 ± 0.4, PCa/PNamin = 14.0 ± 1.6 and PCa/PNamax = 21.7 ± 1.7 (n = 12). The extent
of shift in divalent cation selectivity with cGMP, however,
was less in cones than in rods. The decrease in Ca2+ permeability between saturating and zero cGMP (PCa/
PNamax and PCa/PNamin) was 1.55 ± 0.21 (n = 12). Thus,
in intact, dark-adapted photoreceptor cells, where only
~3% of the channels are open, PCa/PNa in cones can be
expected to be 7.41 ± 0.85-fold larger than in rods.
|
Monovalent Cation Selectivity Is Independent of cGMP in Channels of Cones
We tested whether in channels of cones, like those of
rods, the selectivity among monovalent inorganic cations was independent of cGMP. We studied Cs+ and
NH4+ selectivities relative to Na+ using cGMP concentration ramps under biionic solutions of 150 mM Cs+/
Na+ or 150 mM NH4+/Na+ (Fig. 13). At saturating
cGMP concentrations, the reversal potentials were 4.2 ± 0.53 (n = 5) for Cs+ and 16.4 ± 1.4 mV (n = 6) for
NH4+. Eq. 1 yields PCs/PNa = 0.87 ± 0.02 and PNH4/
PNa = 1.94 ± 0.11, similar values to those previously
reported by others (Picones and Korenbrot, 1992
;
Haynes, 1995
). We found no changes in the reversal potentials at different cGMP concentrations. Thus, in cone channels, gating modifies the selectivity for divalent but not monovalent cations.
|
![]() |
DISCUSSION |
---|
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---|
We report that cyclic nucleotide-gated (CNG) channels in both rod and cone retinal photoreceptors
change their relative Ca2+ to Na+ selectivity as a function of cGMP concentration. The linkage between selectivity and channel activity is specific for divalent cations and is not observed when the selectivity among inorganic or organic monovalent cations is explored.
The dependence on cGMP concentration of the
changes in relative ion selectivity is well described by a
Hill equation, the same one that describes the dependence of probability of channel opening on the nucleotide concentration. The coupling of ion selectivity
and gating is a feature of heteromeric recombinant rod
channels formed by and
subunits, but is absent in
homomeric channels formed by
subunits alone.
Two alternative molecular mechanisms could explain our macroscopic findings of the effect of cGMP on ion selectivity: (a) two or more types of CNG channels exist that differ in their ion selectivity or (b) only one type of channel exists that exhibits two or more conductance states, each of different ion selectivity. If two distinct channels exist, then one must have a high sensitivity to cGMP and low PCa/PNa, while the other must have a lower sensitivity to cGMP and higher PCa/PNa. Thus, at saturating cGMP concentrations, both channel types would be open and the selectivity would be the weighted sum of the selectivities of each type. As cGMP concentration declines, the low sensitivity channel would close, and the selectivity of the high sensitivity channel would define the selectivity observed experimentally. If a single molecular type of channel exists with multiple conductance states, than the probability of occupying different conductance states must be cGMP dependent, and the conductance states that exist primarily at low cGMP concentrations should have a much lower Ca2+ permeability than states that exist at high cGMP concentrations.
In intact rod photoreceptors, there exist two molecularly distinct types of cGMP-gated channels that differ
in their kinetic properties (Torre et al., 1992). In the
inner segment membrane, ~1 of 20 CNG channels exhibit slow kinetics that are similar to those of recombinant channels composed of
subunits alone (Torre et al., 1992
). The remaining channels, in contrast, exhibit rapid flickering similar to that observed in recombinant
channels (Torre et al., 1992
; Chen et al.,
1993
) and in channels of the outer segment (Taylor
and Baylor, 1995
). We do not think that the existence
of these two kinetically distinct types of channels underlie the macroscopic behavior we report here since:
(a) the channels are located in the inner segment of
the cell and their copy number is variable, yet we observe cGMP-dependent permeability changes on every
patch isolated from the outer segment alone; and (b)
the two known channel types have identical sensitivity to cGMP, yet our observations demand that the channel types differ in their sensitivity to cGMP.
Biochemical experiments have previously suggested
that two types of cGMP-gated channels might exist in
rod outer segments. Experiments on purified bovine
rod outer segment membrane vesicles revealed two
cGMP-dependent components with different Ca2+
efflux kinetics (Koch et al., 1987). In these findings,
however, the high Ca2+ permeability component has a
high affinity for cGMP. This behavior is contrary to our
electrophysiological findings. Moreover, additional evidence suggests that the two kinetic components observed in the biochemical studies likely reflect the existence of two types of membrane vesicles, those with and
those without Na+-Ca2+,K+ exchangers, rather than two
different types of cGMP-gated channels (Schnetkamp,
1987
).
Modulation might give rise to the presence of populations of functionally distinct channels in the same
patch. Recordings of cGMP-dependent currents in
both photoreceptor and oocyte membranes show a
high variability in the absolute value of K1/2 from patch to patch and over time in the same patch, a fact that
may reflect modulation, perhaps due to channel phosphorylation (Gordon et al., 1992; Ruiz et al., 1999
).
Also, an endogenous modulator, partially mimicked by
calmodulin, is known to modify K1/2 in both rods (Hsu
and Molday, 1994
; Gordon et al., 1995
) and cones
(Hackos and Korenbrot, 1997
; Rebrik and Korenbrot,
1998
). To explain the data presented here, known
modulation of the CNG channels would have to affect
both the affinity for cGMP and the divalent cation selectivity. In the case of calmodulin or the calmodulin-like endogenous modulator, PCa/PNa of the channels
in the presence and absence of the modulator is the
same (Hackos and Korenbrot, 1997
), and thus such
modulation cannot explain the cGMP- dependent changes
in selectivity.
Any form of modulation that changes both the cGMP affinity and divalent cation selectivity should give rise to two observable features in our data. (a) The extent of modulation should be measurable by observing either the maximal PCa/PNa or K1/2. Thus, maximal PCa/ PNa (that measured at saturating cGMP) should vary from patch to patch, and over time in the same patch, just as K1/2 varies. (b) Patches with low K1/2 would be expected to have low maximal PCa/PNa values and vice versa. While K1/2, and to a lesser extent maximal PCa/ PNa, vary from patch to patch, we did not find any correlation between K1/2 and maximal PCa/PNa, making it unlikely that modulation or in fact any multiple channel mechanism could explain our findings.
If multiple populations of channels with different
cGMP sensitivity and ionic selectivity do not coexist in
patches of CNG channels, than there must be a more
direct mechanism involved. A direct functional linkage
between ion selectivity and gating has been previously
observed in both voltage- and ligand-gated channels.
In the presence of biionic solutions of monovalent
cations, N-methyl-D-aspartate-gated currents do not
exhibit a single reversal potential, and current fluctuations do not disappear at the reversal voltage (Schneggenburger and Ascher, 1997). Single channel recordings demonstrate that this macroscopic behavior reflects the
existence of at least two subconductance states that
differ in their ion selectivity (Schneggenburger and
Ascher, 1997
). Shaker K+ channels exhibit at least two
subconductance states that differ in their monovalent
cation selectivity (Zheng and Sigworth, 1997
). Our observations could be explained if the open pore of a single channel had several possible structural states that
differ in divalent cation permeability. Each of these
open states might also correspond to the well documented subconductance states of the CNG channels.
The presence of multiple conductance states in CNG
channels was recognized in the first published single
channel recordings (Haynes et al., 1986; Zimmerman
and Baylor, 1986
), but only later analysis focused on
these features. In studies of bovine rod membrane vesicles incorporated into lipid bilayers, Ildefonse and Bennett (1991)
observed several single channel conductance states and proposed that sequential binding of
four cGMP molecules correspond to the opening of
four discrete conductance levels. Taylor and Baylor
(1995)
observed subconductance levels in single channel recordings from tiger salamander rods and reported that the fraction of time spent in the subconductance level decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening of partially liganded channels. Since cGMP-gated channels are tetrameric with four cyclic nucleotide binding sites (Liu et al., 1996
), the subconductance levels have generally been interpreted as representing distinct states of one, two, or three bound
cGMP molecules. Ruiz and Karpen (1997)
, however,
have suggested that this interpretation is incorrect. Using a photocross-linkable cGMP analogue, they locked
single channels formed from
subunits in a specific
ligand-bound state. Their results indicate that these
channels do not have significant probability of opening until at least three ligands are bound and that the number of bound cGMP molecules alters the probability of
occupying a particular open state, but does not define
which state is occupied. Triply liganded channels display two strong subconductance states in addition to
the fully open state, while the fully liganded channel mainly occupies only the fully open conductance state.
Consistent with these findings, we suggest that the macroscopic effect of cGMP on ion selectivity may reflect
the existence of at least two conductance states each of
distinct ion selectivity. At low cGMP, the prevalent state
is predicted to be of lower PCa/PNa than the state
prevalent at high cGMP concentration.
What types of pore structural changes might give rise
to the subconductance states and the cGMP-dependent
permeability changes observed here? Sun et al. (1996)
have shown in a series of cysteine accessibility studies
that the CNG channel pore may undergo large structural changes during gating and may in fact be the gate
itself. Particularly intriguing is the finding that tetracaine binds to the pore, depending on whether it is
open or closed, by forming a salt bridge with the E363
residue within the pore (Fodor et al., 1997
). The fact
that tetracaine does not bind to the open channel may
indicate that a conformational change within the pore alters either the position of E363 or its accessibility to
tetracaine. Since E363 is critical in determining the divalent cation block and permeation in the cGMP-gated
channel (Root and MacKinnon, 1993; Eismann et al.,
1994
), and since this residue may change its position or
accessibility in the course of gating (Fodor et al., 1997
),
it is then possible that this structural change may also
contribute to linking changes in gating with changes in
divalent cation selectivity. If changes in open pore
structure occur, these are not reflected in the steric dimensions of the pore since the selectivity for monovalent
organic cations, a simple test of steric hindrance in these
channels (Picco and Menini, 1993
), is unaffected by
cGMP. Changes not within, but around the mouth of the
pore might also affect selectivity (Seifert et al., 1999
).
The proposition that cGMP controls the prevalence of conducting state of different ion selectivity might be tested experimentally in single channel studies, but not with channels formed from recombinant alpha subunits alone since these channels do not exhibit cGMP-dependent changes in selectivity. On theoretical grounds, however, a simple model of two conducting states (triply liganded and fully saturated), each with different divalent cation selectivities, cannot fully explain our results. This is because a model of two conducting states with four cGMP binding sites can be shown to predict that changes in divalent cation selectivity as a function of cGMP should show little, if any, cooperativity when compared with the cooperativity of the current-cGMP relationship. Contrary to this expectation, we have found that the dependence of changes of PCa/PNa on cGMP is the same as the dependence of current on cGMP. Thus, a more complex mechanism must be at play. Resolving whether cGMP changes ion selectivity by affecting the structure of the pore or by changing the probability of opening of subconductance states of differing ion selectivity must await further experimental work.
The stoichiometry of and
subunits in the cyclic
nucleotide-gated channels appears to be 2:2 in channels of olfactory neurons (Shapiro and Zagotta, 1998
).
The photoreceptor
subunit, while itself unable to
form functional channels (Chen et al., 1993
), confers
several functional properties to the channel that are absent in channels formed from
subunits alone. These
properties include: (a) the ability to flicker rapidly
(Torre et al., 1992
), (b) the Ca2+-dependent modulation of sensitivity to cGMP mediated by calmodulin (Chen et al., 1994
), (c) increased sensitivity to diltiazem (Chen et al., 1993
), and (d) reduced sensitivity
to Ca2+ block (Korschen et al., 1995
). We now add the
dependence on cGMP of divalent cation selectivity and
channel block.
Physiological Implications of the Results
Under physiological conditions, in both rods (Hestrin
and Korenbrot, 1987; Cameron and Pugh, 1990
) and
cones (Cobbs et al., 1985
; Miller and Korenbrot, 1993
),
the probability of opening of the cGMP-gated channels
ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination. That is, in the intact photoreceptor, channels spend
nearly all their time exposed to very low concentrations
of cGMP. Since the Ca2+ permeability reaches its minimum value at low cGMP concentrations, we would not
expect the Ca2+ permeability to change significantly in
the course of the normal photoresponse. However, if
the channel's Ca2+ selectivity changes with cGMP in
other cells, where the operating range of changes in cytoplasmic is larger than in photoreceptors, then modulation of Ca2+ fluxes by cGMP (or cAMP) should be
considered as a potentially important physiological modulation.
Cone cGMP-gated channels are more permeable to
Ca2+ than those in rods (Frings et al., 1995; Haynes,
1995
; Picones and Korenbrot, 1995
). At saturating
cGMP concentrations, we find that channels from
striped bass cones are ~3.3-fold more permeable to
Ca2+ than those of tiger salamander rods, a result consistent with previous measurements (Picones and Korenbrot, 1995
). This difference is even larger at physiologically relevant cGMP concentrations. At the cytoplasmic cGMP concentrations expected in dark adapted cells, PCa/PNa in cone channels is ~7.4-fold greater
than that in rods. The physiologically significant parameter, however, is not the difference in the values of
PCa/PNa, but in the fraction of the ionic current carried by Ca2+ in rods and cones.
![]() |
FOOTNOTES |
---|
Address correspondence to Juan I. Korenbrot, Department of Physiology, School of Medicine, Box 0444, University of California at San Francisco, San Francisco, CA 94143. Fax: 415-476-4929; E-mail: juan{at}itsa.ucsf.edu
Original version received 26 March 1999 and accepted version received 21 April 1999.
In loving memory of Christine Mirzayan. ![]() |
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