From the * Graduate Program in Biophysics, and Department of Physiology, School of Medicine, University of California at San Francisco, San Francisco, California 94143
To investigate modulation of the activation of cGMP-gated ion channels in cone photoreceptors, we measured currents in membrane patches detached from the outer segments of single cones isolated from striped bass retina. The sensitivity of these channels to activation by cGMP depends on the history of exposure to divalent cations of the membrane's cytoplasmic surface. In patches maintained in 20 µM Ca++ and 100 µM Mg++ after excision, the current amplitude dependence on cGMP is well described by a Hill equation with average values of K1/2, the concentration necessary to activate half the maximal current, of 86 µM and a cooperativity index, n, of 2.57. Exposing the patch to a solution free of divalent cations irreversibly increases the cGMP sensitivity; the average value of K1/2 shifts to 58.8 µM and n shifts to 1.8. Changes in cGMP sensitivity do not affect other functional parameters of the ion channels, such as the interaction and permeation of mono- and divalent cations. Modulation of cGMP activation depends on the action of an endogenous factor that progressively dissociates from the channel as Ca++ concentration is lowered below 1 µM. The activity of the endogenous modulator is not well mimicked by exogenously added calmodulin, although this protein competes with the endogenous modulator for a common binding site. Thus, the modulation of cGMP affinity in cones depends on the activity of an unidentified molecule that may not be calmodulin.
Key words: retina; phototransduction; calmodulin; rod photoreceptor; fishRod and cone photoreceptors and olfactory sensory
neurons respond to their appropriate stimuli with changes
in membrane conductance that reflect the activity of cyclic nucleotide-gated ion channels (Fesenko et al., 1985;
Nakamura and Gold, 1987
; Haynes and Yau, 1990
). In
these cells, the stimuli activate enzymatic cascades that
ultimately change the cytoplasmic concentration of either cGMP (photoreceptors) (reviewed by Hurley, 1992
;
Pugh and Lamb, 1993
; Baylor, 1996
) or cAMP (olfactory neurons) (reviewed by Reed, 1992
). Because of
similarities in primary structure, the cyclic nucleotide-
gated channels in the sensory cells have been recognized to be members of the same gene family (for review see
Zagotta and Siegelbaum, 1996
; Finn et al., 1996
; Yau
and Chen, 1995
). Despite their general structural similarities, the chemical specificity and affinity of the ligand
binding sites differ among channels of the various sensory cell types (Scott et al., 1996
). For example, the
channels in retinal photoreceptors have a 20- to 50-fold
higher affinity for cGMP than cAMP, whereas native
channels of olfactory neurons have nearly the same affinity for both nucleotides (see review in Zagotta and
Siegelbaum, 1996
). Even in the same receptor cell type,
the nucleotide affinity of the channels varies from species to species (reviewed in Zagotta and Siegelbaum,
1996
), presumably optimized to match the nucleotide
cytoplasmic concentration in the cell expressing that
specific channel. Furthermore, recent experiments have
shown that the ligand affinity of the channels is not
static, but changes as a function of Ca++ concentration
(reviewed in Molday, 1996
). The physiological significance of this Ca++ dependence is not fully understood.
However, it is likely to play an important role in the adaptation of the cell's response to changing background
levels of stimulation (Kurahashi and Menini, 1997
).
The extent to which Ca++ modulates the cGMP activation differs markedly between olfactory and rod photoreceptor channels. In rat olfactory neurons, for example, affinity for cAMP decreases over 100-fold when
Ca++ is elevated to 200 µM (Chen and Yau, 1994; Liu et
al., 1994
; Balasubramanian et al., 1996
). Affinity is measured by the value of K1/2, the concentration necessary
to activate half the maximum ligand-gated current. In
rod photoreceptors, the Ca++ modulation of K1/2 has
been studied in isolated membrane vesicles (Bauer,
1996
), detached membrane patches (Gordon et al.,
1995
), and truncated outer segments (Nakatani et al.,
1995
; Sagoo and Lagnado, 1996
). In all these preparations, the K1/2 for cGMP activation shifts ~1.5-fold with
a Ca++ dependence that is half maximal at ~50 nM.
Because this modulation is irreversibly lost in patches
and truncated rods after exposure to solutions free of
divalent cations, it has been presumed to arise from the
action of a soluble, endogenous modulator (Gordon et
al., 1995
; Nakatani et al., 1995
; Sagoo and Lagnado,
1996
; Bauer, 1996
). The molecular identity of this endogenous modulator in rods is not fully resolved, but
the possibility that it is calmodulin has been addressed
in a number of recent experiments.
Hsu and Molday (1993) first demonstrated that calmodulin can cause a Ca++-dependent modulation of
K1/2 in rods. In a manner similar to the endogenous
modulator, the maximum Ca++/calmodulin-dependent
shift in K1/2 is ~1.5-fold (Gordon et al., 1995
; Kosolapov and Bobkov, 1996
; Bauer, 1996
). The Ca++ dependence of the shift in K1/2 in the presence of added
calmodulin has a half-maximum value of ~50 nM in
some studies (Hsu and Molday, 1993
; Bauer, 1996
) and
450 nM in others (Kosolapov and Bobkov, 1996
).
These values, however, cannot be compared with those
characteristic of the endogenous modulator because
they change with the calmodulin concentration used in
the experiments (Bauer, 1996
). Gordon et al. (1995)
and Bauer (1996)
have directly investigated whether the
endogenous factor and calmodulin are one and the
same. In membrane vesicles, there are no recognizable
differences in the function of the two molecules (Bauer,
1996
). However, in membrane patches, the function of
both molecules was found to differ in several respects,
suggesting that the modulator in rods may differ from
calmodulin (Gordon et al., 1995
). In truncated rods,
pharmacological blockers of calmodulin do not alter the
Ca++-dependent modulation of K1/2, nor does added
calmodulin confer modulation (Sagoo and Lagnado,
1996
). Finally, Haynes and Stotz (1997)
have found that
added calmodulin can modulate K1/2 in rod membrane
patches, but not in those of cones from the same species.
The cyclic nucleotide-gated channels of cone outer
segments are structurally homologous to those of rods
(Bonigk et al., 1993; Weyand et al., 1994
), yet their
functional properties differ in subtle but important
ways. These functional differences may contribute to
explain the differences in transduction between the
two receptor types (Korenbrot, 1995
). The K1/2 of
cGMP binding is higher in cones (Haynes and Yau,
1990
; Picones and Korenbrot, 1992
) than in rods
(Karpen et al., 1988
; Zimmerman and Baylor, 1992
;
Haynes and Stotz, 1997
); the energy of interaction of
cations such as Na+ and Li+ with the channel is higher
in cones (Picones and Korenbrot, 1992
; Haynes, 1995
)
than in rods (Menini, 1990
; Furman and Tanaka, 1990
; Zimmerman and Baylor, 1992
), and the blocking effect
of l-cis-diltiazem is also different (Haynes, 1992
). Of singular functional significance is the fact that the permeability and interaction of Ca++ with the channels differs
between the two photoreceptor types (Korenbrot, 1995
).
In particular, the permeability of Ca++ relative to Na+
is higher in cone than in rod channels (Picones and
Korenbrot, 1995
). This difference is also observed in
recombinant channels formed by cone or rod
subunits alone (Frings et al., 1995
). Whether there exists a
calcium-dependent modulation of ligand binding in
cone channels and what features this modulation might have has not been previously investigated. We report
here that in membrane patches of cone outer segments, as in those of rods, there exists a Ca++-dependent modulation of cGMP activation. This modulation
depends on the activity of an endogenous modulator.
We further contrast the properties of rod and cone
channels with respect to the modulatory effect of
added calmodulin. In cones, the endogenous modulator is not well mimicked by calmodulin.
Materials
We obtained striped bass (Morone saxitilis) from Professional Aquaculture Services (Chico, CA) and tiger salamanders (Ambystoma tigrinum) from Charles Sullivan (Memphis, TN). We received cGMP, 8-Br-cGMP, and calmodulin (bovine brain phosphodiesterase activator) from Sigma Chemical Co. (St. Louis, MO).
Photoreceptor Isolation
Methods of cell isolation are described in detail elsewhere
(Miller and Korenbrot, 1993b, 1994
). Animals were dark adapted and retinas were isolated under infrared illumination. Single cones were obtained by mechanical dissociation of striped bass retinas maintained in a Ringer's solution consisting of (mM): 143 NaCl, 2.5 KCl, 5 NaHCO3, 1 Na2HPO4, 1 CaCl2, 1 MgCl2, 5 pyruvate, 10 HEPES, pH 7.5, osmotic pressure 309 mOsM. Rods were
isolated by mechanical dissociation of tiger salamander retinas
maintained in a Ringer's solution composed of (mM): 100 NaCl,
2 KCl, 5 NaHCO3, 1 Na2HPO4, 1 CaCl2, 1 MgCl2, 5 pyruvate, 10 HEPES, pH 7.4, 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 stage of an upright microscope equipped
with DIC optics and operated under visible light. A suspension of
photoreceptors in pyruvate-Ringer's was placed on the coverslip
and the cells were allowed to settle down and attach for 5 min.
The bath solution was then exchanged with a Ringer's solution of
the same composition, but in which pyruvate was isosmotically replaced with glucose.
The recording chamber consisted of two side-by-side compartments. Cells were held in one compartment that was continuously perfused with 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. After forming a giga-seal and detaching the membrane
fragment, we moved the electrode under the solution surface
from the compartment containing the intact cells to the smaller
compartment. The two compartments were then separated by
the movable barrier and the tip of the electrode was placed
within 100 µm of the opening of a 300-µm diameter glass capillary. We used this capillary and a rotary valve to deliver selected
test solutions onto the cytoplasmic (outside) surface of the membrane patch.
The speed of change in membrane current in response to
changes in test solutions varied from patch to patch. This variability likely reflects differences among patches in the accessibility of
their cytoplasmic surface to the bath solution. In our experiments, after each solution change, we monitored membrane current by
repeated presentation of voltage steps and waited for the current
to reach a stationary amplitude. We present and analyze here only
currents in this stationary condition (except for Fig. 5).
Ionic Solutions
In studies of both cone and rod membranes, we filled the tight-seal electrodes with the same solution (mM): 157 NaCl, 5 EGTA, 5 EDTA, 10 Hepes, adjusted with NaOH to pH 7.5, osmotic pressure 305 mOsM. Free Ca++ concentration in this solution was
10
10 M and total Na+ concentration was 167 mM. In all studies, the initial composition of the solution in the small compartment into which we moved the electrode was 157 mM NaCl, 10 mM Hepes, adjusted with NaOH to pH 7.5, and 20 µM free Ca++
(total 7.637), 100 µM free Mg++ (total 2) with 10 mM HEDTA.
We elected to use these free divalent cation concentrations because they are sufficiently low to not block the cGMP-gated conductance (Picones and Korenbrot, 1995
), yet sufficiently high to
saturate the Ca++ dependence of the phenomena studied here.
We used a titration method to produce solutions with free calcium concentrations between 10 nM and 20 µM (Williams and
Fay, 1990). This method yields accurate free calcium concentrations without the need to weigh with extreme precision chemicals of known purity. This is particularly important because 100%
pure Ca++ buffering agents cannot be obtained commercially
(Miller and Smith, 1984
). Briefly, a solution containing (mM)
157 NaCl, 10 HEPES, 10 HEDTA, adjusted with NaOH to pH 7.5, was divided into two parts. One part (solution A) had no added
calcium. Into the other part (solution B), CaCl2 was added to 9 mM and the pH readjusted to 7.8. A small sample of this solution
was then titrated with 100 mM CaCl2 while monitoring its pH.
The amount of CaCl2 necessary to fully titrate HEDTA, that is, to
obtain pure CaHEDTA at pH 7.5, was determined from this titration. The appropriate amount of CaCl2 was then added to the remaining solution B. Solutions A and B were mixed to obtain different free calcium concentrations (Table I). Volumes were calculated (EqCal; BioSoft, Cambridge, UK) using published values
for HEDTA binding constants (Martell and Smith, 1974
). The
pH was adjusted to 7.5 and solutions stored at 4°C in plastic containers for up to 1 wk. Solutions containing both calcium and
magnesium (at concentrations of 20 and 100 µM free, respectively) were made by adding to solution A appropriate amounts
of stocks of CaCl2 and MgCl2. The concentration of divalent cations in these stocks were calibrated by measuring their osmolality.
Table I. Solutions of Defined Free Ca++ Concentration Using HEDTA |
In many of the experiments reported here, we tested the electrical properties of the same membrane patch before and after removing divalent cations. Divalent cations were removed by exposing the cytoplasmic (outside) surface of the patch to a solution composed of (mM): 157 NaCl, 10 HEPES, 5 EDTA, 5 EGTA, adjusted with NaOH to pH 7.5. We refer to this solution in the following text simply as the EDTA/EGTA solution.
Electrical Recordings
Tight-seal electrodes were made from aluminosilicate glass
(1724; Corning Glass Works, Corning, NY) (1.5 × 1.0 mm, o.d. × i.d.). We measured membrane currents under voltage clamp at
room temperature (19-21°C) with a patch-clamp amplifier
(#8900; Dagan Instruments, Minneapolis, MN). Analog signals
were low pass filtered below 2.5 kHz with an eight pole Bessel filter and were digitized on line at 6 kHz (FastLab; Indec, Capitola,
CA). Membrane voltage was normally held at 0 mV and membrane currents were activated either by 110-ms long step changes
to 40 mV or with a continuous voltage ramp that swept between
70 and +70 mV at a rate of 228 mV/s. In measurements with
voltage ramps, the voltage was stepped from 0 to
70 mV and
held at that value for 200 ms before applying the voltage ramp.
This was necessary because in the presence of divalent cations
there is a time-dependent change in membrane current amplitude on switching from 0 to
70 mV. This time-dependent change is due to the well characterized voltage-dependent channel block by divalent cations (in rods, Zimmerman and Baylor,
1992
; in cones, Picones and Korenbrot, 1995
). To generate current-voltage (I-V)1 curves, the voltage ramp was swept in four
successive trials and the currents were signal averaged. Between
voltage ramp trials, membrane voltage was held at 0 mV for 1.2 s.
As usual, outward currents are positive and the extracellular
membrane surface is defined as ground.
We began every experiment by determining the current amplitude before and after adding saturating cGMP concentrations.
We frequently found (>50%) that membrane patches did not respond initially to the ligand. In many instances, these patches became responsive after rapidly moving the electrode tip across the
air-water interface. It is likely, therefore, that unresponsive
patches had formed closed vesicles that opened upon crossing
the air-water interface (Horn and Patlak, 1980). We only analyzed data measured in patches in which the amplitude of the
current generated with saturating cGMP concentrations and the
leakage current measured in the absence of cGMP did not
change by >10% over the course of the entire experiment. Functions were fit to experimental data by least square minimization
algorithms (Origin; MicroCal Software, Northampton, MA). Experimental errors are presented as standard deviations.
The K1/2 of the cGMP-gated Currents in Cones Is Modulated by Divalent Cations
We investigated cGMP-dependent currents in inside-out
patches detached from the plasma membrane of retinal
cone outer segments. These patches contain only cGMP-gated ion channels (Miller and Korenbrot, 1993a). The
dependence of current amplitude on cGMP concentration is well described by the Hill equation (Fig. 1):
![]() |
(1) |
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 value of Imax, and n is a parameter that reflects the cooperative interaction of cGMP molecules in activating the membrane current.
The cGMP dependence of the membrane current depends on the history of exposure of the membrane
patch to divalent cations. In Fig. 1, we present cGMP-dependent currents measured in a cone membrane
patch in response to 40 mV voltage steps and in the presence of 20 µM Ca++ and 100 µM Mg++. Currents
were measured in the same patch both before and after exposure to the EDTA/EGTA solution (Fig. 1, A and
B). Also illustrated is the dependence of current amplitude on cGMP concentration (Fig. 1 C). The data
points measured both before and after exposing the
patch to EDTA/EGTA were well fit by Eq. 1, but the values of K1/2 and n are different. The average value of K1/2
shifted from 86.1 ± 18 to 58.8 ± 19 µM upon exposure
to EDTA/EGTA. The average value of n shifted from
2.57 ± 0.34 to 1.80 ± 0.23 (Table II). Experimental
data were included in these averages only if cGMP activation was measured in the same patch before and after exposure to the EDTA/EGTA solution. The shift in K1/2
and n reflect a divalent cation-dependent mechanism
that modulates the channel's sensitivity to the cyclic nucleotide.
Table II.
Modulation of Cyclic Nucleotide-gated Nucleotide-gated Currents in Membrane Patches of Rods and Cone *, |
To compare the functional properties of rod and
cone cGMP-gated channels, we also investigated the
Ca++-dependent modulation in patches from the rod
outer segments of tiger salamanders (Fig. 1 D). In these
membranes, in the presence of 20 µM Ca++ and 100 µM Mg++, K1/2 for cGMP was 41.1 ± 7 µM and n was
2.58 ± 0.43 before exposure to EDTA/EGTA, and K1/2
was 27.5 ± 6.2 µM and n to 1.97 ± 0.28 afterwards (Table II). Thus, in tiger salamander rods K1/2 shifts by
~1.5-fold, similar to findings in frog rods (Gordon et
al., 1995). The fractional change in K1/2 is thus similar
in rod and cone membrane patches.
Modulation of K1/2 Is Not an Artifact Due to Phosphodiesterase Activity in the Membrane Patch
Detached outer segment patches can be structurally
complex and may include not only plasma membrane
but fragments of disc membranes containing phosphodiesterase (PDE) activity (Ertel, 1990). This may be
a particular concern in cone outer segments, where
disc and plasma membranes are continuous. The presence of PDE in the patch can lead to the artifactual appearance of variable cGMP titration curves (Ertel, 1990
).
We tested whether this might be a possible source of
experimental artifacts in cone membrane patches by
comparing cGMP activation curves in the presence and absence of 0.5 mM IBMX (3-isobutyl-1-methylxanthine),
a saturating concentration of this effective cone PDE inhibitor (Gillespie and Beavo, 1989
). IBMX had no effect
whatsoever on the titration curves (data not shown).
We confirmed further that the modulation of K1/2 is
not an artifact due to the effect of PDE by measuring
the properties of membrane currents activated by 8-Br-cGMP. This cGMP analog activates the channels but is
not efficiently hydrolyzed by the photoreceptor PDE
(Zimmerman et al., 1985). Fig. 2 illustrates currents activated by 8-Br-cGMP and measured in the same patch
in the presence of 20 µM Ca++ and 100 µM Mg++ before and after exposure to EDTA/EGTA. As with cGMP
activation, the dependence of current amplitude on
8-Br-cGMP was well described by Eq. 1 and removal of
divalent cations shifted K1/2 and n to lower values (Table I). Thus, modulation of the cGMP-gated currents does not reflect the activity of PDE.
Ion Permeation and Selectivity Are Not Different in the Two States of Ligand Affinity
We investigated whether the state of modulation affects
other functional properties of the cGMP-gated currents. The shape of the I-V curve reflects, according to
Eyring rate theory, the energy of interaction between
permeant cations and their binding sites within the
channel (Alvarez et al., 1992). The success of this theoretical analysis is measured by the ability to predict the
shape of the I-V curves measured under various ionic
conditions. The I-V curves of the cGMP-gated channels
of both cones (Picones and Korenbrot, 1992
; Haynes,
1995
) and rods (Zimmerman and Baylor, 1992
) can be
predicted using Eyring rate theory, assuming the energy profile across the channels includes one binding
site asymmetrically located within the membrane. If the
interaction between permeant cations and the channel
is affected by the state of modulation, then the shape of
the I-V curve should change.
We compared in detail I-V curves measured in the
same cone membrane patch before and after exposure
to EDTA/EGTA. Fig. 3 illustrates membrane currents
measured under symmetrical NaCl solutions with 20 µM Ca++ and 100 µM Mg++ on the cytoplasmic membrane surface and no divalent cations on the extracellular surface. Currents were measured in the presence of
various cGMP concentrations in the range between 40 µM and 1 mM, and activated with a continuous voltage
ramp between 70 and +70 mV. The I-V curves were
nonlinear under all cGMP concentrations tested (Fig.
3). The nonlinearity is generated both by the voltage dependence of cGMP binding and the voltage dependence
of divalent block (reviewed in Yau and Baylor, 1989
). To
analyze the I-V curves, we determined the voltage dependence of the binding curve for cGMP by fitting Eq. 1 to
the current measured at every voltage between
70 and
+70 mV. We found n not to change significantly with
voltage either before or after exposure to EDTA/EGTA.
K1/2, on the other hand, was voltage dependent, but the
form of the voltage dependency was quantitatively the
same before and after exposure to EDTA/EGTA (Fig.
3). Thus, the I-V curves are not affected by the state of
modulation. We obtained similar results in five other
cone patches and four rod patches. Hence, the energy of interaction between cations and the channels, in both
rods and cones, does not appear to be affected by the
state of channel modulation.
We analyzed the relative Ca++ to Na+ permeabilities
of the channels (PCa/PNa) in the two states of ligand
affinity. This is a physiologically important feature since
PCa/PNa differs in channels of rods and cones (Picones and Korenbrot, 1995; Frings et al., 1995
). To determine the value of PCa/PNa, we measured I-V curves (with voltage ramps) under saturating cGMP concentrations (1 mM). The concentration of NaCl was symmetric across the membrane, and we imposed CaCl2
concentration gradients. The extracellular membrane surface was free of Ca++ and the cytoplasmic membrane surface was exposed to concentrations of 5 or 10 mM. The same patch was then exposed to the EDTA/
EGTA solution, and the current measurement repeated. Typical results are illustrated in Fig. 4. The reversal potential shifted towards negative values as the
cytoplasmic Ca++ concentration increased, indicating
that the channels are more permeable to Ca++ than to
Na+. The magnitude of the shift was the same before
and after exposure to EDTA/EGTA. Thus, the state of
modulation does not affect the ion selectivity of the
cone channels. We obtained the same results in three
other membrane patches. The values of PCa/PNa, calculated from the shift in reversal voltage (Lewis, 1979
),
are similar to those we have previously reported (Picones and Korenbrot, 1995
).
Effects of Ca++ on the Endogenous Modulation of cGMP Affinity
The removal of both Ca++ and Mg++ results in an irreversible shift of cGMP affinity. We investigated whether this shift was specific for either cation by testing whether the cGMP affinity shifted when only one of the two cations was removed. Removal of Mg++ alone did not shift the ligand affinity. Removal of Ca++ alone shifted the cGMP activation curve just as when both cations were removed, but the rate of this shift was slower than that caused by the simultaneous removal of both cations.
We explored the effects of varying Ca++ concentration on the shift in cGMP affinity. We measured the current activated by 60 µM cGMP in the presence of 20 µM Ca++ and 100 µM Mg++, exposed the patch for 60 s to solutions free of cGMP with the same Mg++ but progressively lower Ca++ concentrations, and then repeated the current measurement in the 20 µM Ca++, 100 µM Mg++ solution with cGMP. The current was unaffected at concentrations as low as 1 µM Ca++. Below this concentration, current amplitude increased as Ca++ concentration declined (Fig. 5). The increase in current amplitude at a fixed, nonsaturating cGMP concentration reflects shifts in K1/2 and n to lower values. The current continued to increase, but even at 100 nM Ca++ the current increase was not maximal. Addition of EDTA/EGTA achieved the maximum current enhancement and additional washes in EDTA/EGTA caused no further current enhancement (Fig. 5). We made the same observations in six other patches. Because the shift in K1/2 is irreversible, the experimental data do not reflect stationary conditions and, therefore, cannot be quantitatively analyzed as equilibrium dose- response data. Nonetheless, the results indicate that in detached patches, cGMP affinity is specifically modulated by Ca++ over a concentration range limited by ~1 µM at its upper end.
The Effects of Exogenous Calmodulin
In rods, the Ca++-dependent modulation of ligand affinity has been attributed to the action of an endogenous factor similar, and perhaps identical, to calmodulin. Calmodulin confers Ca++ dependence to the cGMP
activation of the channels with features that are almost
indistinguishable from those of the endogenous modulator (Hsu and Molday, 1993; Gordon et al., 1995
; Bauer,
1996
). We explored the potential role of calmodulin in
the modulation of channels in cone outer segments. In
these experiments, we measured the cGMP concentration dependence of currents in the presence of 20 µM
Ca++ and 100 µM Mg++. The membrane patch was
tested before and after exposure to EDTA/EGTA, and
then again in the continuous presence of calmodulin at
a concentration of 200 nM. This concentration is effective in rod membrane patches (Gordon et al., 1995
,
Haynes and Stotz, 1997
) and is well above the concentration that saturates modulation in rod membrane vesicles (Hsu and Molday, 1993
; Bauer, 1996
). In any event,
the concentration of calmodulin used when testing its
pharmacological action affects the Ca++ dependence
of the phenomenon under study, but not its features at
saturating Ca++ concentrations (see Bauer, 1996
, for detailed mathematical analysis).
Fig. 6 illustrates typical results obtained in both rods
and cones. In patches from both photoreceptor types,
as expected, exposure to EDTA/EGTA lowered the values of K1/2 and n in the cGMP titration curves. The addition of calmodulin in the presence of Ca++ shifted
K1/2 and n back towards their initial values. Whereas K1/2
and n essentially reverted to their starting values in rods, the shift was never fully reversed in cones (Table II).
Thus, channels of cones and rods differ in the effectiveness with which calmodulin in high Ca++ shifts their
sensitivity to activation by cGMP. In cones, then, the
Ca++-dependent regulation of cGMP activation is also
likely to reflect the activity of an endogenous modulator. The modulator, however, may not be calmodulin,
since this protein does not fully mimic the endogenous
function.
Calmodulin Competes with the Endogenous Modulator for Binding to the Channels
To test whether the endogenous modulator in cones and calmodulin share structural features, we tested whether the two compete in their binding to the channel. We investigated the effectiveness of added calmodulin to shift K1/2 or n in the presence and absence of the endogenous modulator. If both molecules bind to the same site, then calmodulin should be without effect when added to the membrane in the presence of the endogenous modulator. That is, for calmodulin to be effective, the endogenous modulator must first be removed from its binding site.
We measured membrane currents with voltage steps
between 0 and 40 mV in 20 µM Ca++ and 100 µM
Mg++ in the presence of 60 µM cGMP. At this cGMP
concentration, lowering K1/2 and n will result in an increase in current amplitude despite an unchanging agonist concentration (see Fig. 1). In the same patch, we
measured currents before and after adding 200 nM
calmodulin. Calmodulin was without effect on current
amplitude in membrane patches maintained in high
Ca++ and Mg++ (Fig. 7 A). As expected, removal of the
endogenous modulator by brief exposure to EDTA/
EGTA shifted K1/2 and n and therefore increased the
current amplitude (Fig. 7 B). After exposure to EDTA/
EGTA, added calmodulin was now able to shift K1/2 towards its starting value (Fig. 7 B). We obtained the
same results in every patch tested with this protocol
(n = 6). Thus, while calmodulin and the endogenous
modulator may not be the same, they appear to compete for a common site on the channels.
Ca++ Dependence of the Calmodulin-mediated Modulation
We investigated whether the channels of rods and
cones differ in their interaction with Ca++/calmodulin
by testing the Ca++ dependence of cGMP activation in
the presence of 200 nM calmodulin. This Ca++ dependence is itself a function of the calmodulin concentration (Bauer, 1996) and therefore it does not inform us
as to what the Ca++ dependence of modulation might
be in the intact cell, if calmodulin were the modulator.
However, it will reflect differences in the energetics of
the modulator's binding site between the channels of
the two cell types.
We measured the Ca++ dependence of currents in
membrane patches of rods and cones in the presence
of fixed concentrations of calmodulin and cGMP. In
each experiment, membrane patches were first exposed to the EDTA/EGTA solution to remove the endogenous modulator. In patches from single cones, we
measured currents generated by 110-ms voltage steps to
40 mV in the presence of 60 µM cGMP and varying
Ca++ concentration between 0 and 20 µM. In the same
patch, we measured the effects of Ca++ first in the absence and then in the presence of 200 nM calmodulin (Fig. 8). In cone membranes, Ca++ in the absence of
calmodulin had a small but reproducible effect on current amplitude. The maximum change in current amplitude between 0 and 20 µM Ca++ in the absence of
calmodulin was ~5%. In the data shown, we subtracted this effect to obtain the effect of added calmodulin
alone. We studied rod channels with the same protocols, except that 20 µM cGMP was used to activate the
channel. Fig. 8 illustrates the Ca++ dependence of current amplitude in the presence of calmodulin in typical
patches from both rod and cone channels.
The experimental data were well fit by the function:
![]() |
(2) |
where I is the current, Izero is the current in the absence
of Ca++, I is the current in the presence of a saturating
Ca++ concentration, [Ca++] is the Ca++ concentration,
K i is the Ca++ concentration at which the current is inhibited by one half and n is a parameter that reflects cooperative interaction of cation binding. This is a modified Hill equation that indicates Ca++ interacts cooperatively with calmodulin to block the current amplitude.
For cones, K i = 366 ± 131 nM, n = 1.6 ± 0.47, and I
/
I zero = 0.72 ± 0.16 (N = 9), while for rods, K i = 679 ± 187 nM, n = 1.81 ± 0.44, and I
/Izero = 0.53 ± 0.13 (N = 11). These values suggest that Ca++/calmodulin
interacts with the cGMP-gated channel of both rods and cones, but the quantitative features of this interaction differ between the two receptor types.
The cGMP-gated ion channels in detached patches
from cone outer segments exhibit a Ca++-dependent
modulation of their affinity for the cyclic nucleotide. The dependence of current amplitude on cyclic nucleotide concentration is described by the Hill equation
(Eq. 1) and modulation is manifested as a decrease in
the apparent binding affinity (K1/2) and cooperativity (n) as the Ca++ concentration is lowered. We will refer
to this as the endogenous modulation of the channel.
Endogenous modulation has been previously reported
for the cGMP-gated channels of rods in bovine membrane vesicles (Bauer, 1996), detached frog outer segment patches (Gordon et al., 1995
), and truncated
outer segments from frogs and tiger salamanders (Nakatani et al., 1995
; Sagoo and Lagnado, 1996
). In general, the features of channel modulation in the intact
cell should not be extrapolated using data from patches
alone. In the case of rods, studies in nearly intact truncated outer segments have demonstrated Ca++-dependent modulation of K1/2 that is quantitatively the same as that measured in detached membrane patches (Table III). The caveat has been introduced, however, that
the modulation in truncated rods, which is measured
under stationary conditions, may underestimate the extent of modulation present in the truly intact cell (Sagoo and Lagnado, 1996
). In the case of cones, modulation measured under stationary conditions in the nearly
intact outer segment is larger in extent than that observed in detached membrane patches, the K1/2 shifts
~4- rather than ~1.5-fold (Rebrik and Korenbrot, 1997
).
Table III. Modulation of cGMP-activated Current in Rod Outer Segments |
The endogenous modulation is Ca++ dependent, but
the quantitative features of this dependence cannot be
fully studied in membrane patches because modulation
is irreversibly lost as Ca++ concentration is reduced
and, therefore, equilibrium dose-response curves cannot be determined. In cone patches, the shift in K1/2 is
observed at concentrations starting at and below 1 µM
Ca++ in the presence of 100 µM Mg++. This differs
from data reported for modulation of channels in rods.
In rod membrane patches, under experimental conditions similar to those we have reported for cones, modulation occurs starting at and below 22 nM (Gordon et
al., 1995). This difference is significant and may suggest that, in cones, channel modulation may play a role
in dim light, when only small changes in Ca++ concentration are expected, whereas in rods it may play a relevant role only in signals generated by relatively bright
light (see Bauer, 1996
). It is important to recognize
that the Ca++ dependence of modulation reported for
other rod preparation
for example, washed bovine
membrane vesicles (Bauer, 1996
) or amphibian truncated rods (Nakatani et al., 1995
, Sagoo and Lagnado,
1996
)
may differ from data in detached patches because in each preparation the conditions of equilibrium between the modulator and the channel may be
different. In the detached membrane patch the effective concentration of unbound modulator is essentially
zero. Therefore, the initial condition (high Ca++) is
not at equilibrium and the modulator is kinetically
"locked" onto the channel. The only information that
can be reliably established is the Ca++ concentration at
which the modulator becomes "unlocked" over a reasonably short time course (60 s).
The features of the interaction between the modulator and the channel in the intact cone photoreceptor and its functional role in transduction and/or adaptation are yet to be specified in detail. Although the magnitude of the modulation, a shift of ~1.5-fold in K1/2 may appear modest, the effect of this modulation on current amplitude can be expected to be large, particularly at low cGMP concentrations. From our experimental results, the change in current expected when Ca++ changes from 1 µM to 10 nM is given by:
![]() |
(3) |
where Ilo and Ihi are the currents at 10 nM and 20 µM
Ca++, respectively, [cGMP] is the ligand concentration,
K lo and K hi are the values for K1/2 and n lo and n hi are
the values for n at 10 nM and 20 µM Ca++, respectively.
Fig. 9 plots Eq. 3 with [cGMP] in units of K hi. Also
shown are data points for currents activated by various
cGMP concentrations and measured in a single cone
patch before and after exposure to the EDTA/EGTA
solution. The concentration of cGMP in the dark can
be expected to be between 0.16× and 0.31× K hi since
only 1-5% of the channels are open in darkness
(Cobbs et al., 1985). Thus, if the modulation of the
channel in the intact cell is similar to that in the patch,
the amplitude of the light-sensitive current could
change by as much as 5- to 10-fold in response to changes in cytoplasmic Ca++ concentration. As Eq. 3 indicates, and Fig. 9 illustrates, the effectiveness of
Ca++ as a modulator increases dramatically as the
cGMP concentration is lowered. Under steady illumination, when cGMP concentration is expected to be lower
than in the dark, the physiological role of Ca++ modulation is likely to be especially significant.
The endogenous modulation of K1/2 in photoreceptor membrane patches is irreversibly lost upon removal
of divalent cations, but can be restored, even if to a limited extent, by calmodulin. These results suggest that
endogenous modulation arises from the activity of a
"calmodulin-like" protein, since calmodulin not only
restores modulation but also competes with the endogenous modulator for the same binding site. As has been
argued in reports of similar phenomena in rod patches,
it is unlikely that Ca++ modulation arises from phosphorylation since test solutions lacked nucleotides, or
from phosphatase activity, because these enzymes are
inhibited by lowering Ca++ (Gordon et al., 1995). Thus,
modulation of both rod and cone channels likely arises
from the activity of an endogenous factor that, like
calmodulin, interacts with the channel in a Ca++-dependent manner: it is bound to the channels at high Ca++,
dissociates from them in the absence of Ca++, and is
then lost to the solution.
The molecular identity of the endogenous modulator is under investigation. Calmodulin exists at high
concentration in the rod outer segment (Kohnken et
al., 1981; Bauer, 1996
). Hsu and Molday (1993)
first reported that calmodulin causes a Ca++-dependent shift
in K1/2 in rod channels of thoroughly washed bovine
outer segment membrane vesicles (see also Hsu and
Molday, 1994
; Bauer, 1996
). Similarly, Ca++/calmodulin shifts the K1/2 of channels in rod outer segment
membrane patches (Gordon et al., 1995
; Kosolapov
and Bobkov, 1996
; Haynes and Stotz, 1997
). Gordon et
al. (1995)
compared the functional properties of the
interaction of the endogenous modulator and calmodulin with the channels in frog rod outer segment membrane patches. Several features of this interaction were
quantitatively different between calmodulin and the
endogenous modulator, and the authors were not convinced the two molecules were the same. In contrast,
Bauer (1996)
, in studies of bovine rod membrane vesicles, did not find quantitative differences between the
endogenous modulator and added calmodulin. Thus,
the endogenous modulator in rods is calmodulin-like,
but it is not possible to confirm that it is calmodulin itself.
In contrast to the relative similarity between calmodulin and the endogenous modulator in studies of isolated rod membranes, added calmodulin was found ineffective in inducing a Ca++-dependent shift of K1/2 in
truncated rods of tiger salamander, from which the endogenous modulator was first removed (Sagoo and Lagnado, 1996). This finding is surprising because calmodulin, independently of whether it is the endogenous
modulator, should have had an effect on the truncated
outer segment current since it modulates the channel
in membrane patches detached from the same cells
(Fig. 6), as well as in patches from frog rods (Gordon et
al., 1995
; Kosolopov and Bobkov, 1996). We do not
have a simple explanation for this experimental puzzle,
but it is possible that calmodulin cannot efficiently gain
access to the channels within the truncated outer segment.
Data are not available on the concentration of calmodulin in cone outer segments, nor its association
with their cGMP-gated channels. In our direct comparison, we found that calmodulin did not quantitatively
mimic the activity of the endogenous factor: calmodulin shifted K1/2 and n to a lesser extent than did the endogenous factor. Indeed, Haynes and Stotz (1997) have
reported that in patches of catfish cone outer segments,
calmodulin entirely failed to modulate K1/2. They did
not, however, explore the properties of a possible endogenous modulator: in their experiments, the initial
condition was to hold the membrane patch in a solution containing EDTA and EGTA to remove any endogenous modulator. Thus, while calmodulin does not appear to modulate K1/2 in catfish cones, whether any
modulation occurs at all is unclear. While it might be
surprising that cones in striped bass exhibit modulation while those in catfish do not, it is possible. This issue should be addressed experimentally.
The failure of bovine calmodulin to fully mimic the
action of the endogenous modulator in striped bass
cones could be due to sequence differences between
bovine and fish calmodulin, rather than the nonidentity between calmodulin and the endogenous modulator. This is not likely, however, since the amino acid sequence of calmodulin is nearly 100% identical among
vertebrates (Friedberg, 1990). While the endogenous
modulator may not be calmodulin in cones, the two
molecules are likely to have structural features in common. Calmodulin is a member of a large family of proteins that contain EF hands, a Ca++-binding structural
motif consisting of a select sequence of ~30 amino acids that fold into a helix-loop-helix pattern (Kretsinger, 1979
; Klee and Vanaman, 1982
). Since the endogenous
modulator binds Ca++ and competes with calmodulin
for binding to the channels, it is probably also a member of this family of Ca++ binding proteins. We have
found, however, that other EF hand-containing proteins expressed in photoreceptors, such as GCAP1 and
GCAP2 (reviewed in Polans et al., 1996
) do not restore
modulation to the channels. Furthermore, these proteins do not compete with calmodulin for binding to
the channels (Hackos, Palczewski, Baehr, and Korenbrot, unpublished observations). Knowledge of the
specificity of interaction with the channel of different
members of the EF hand protein family will be important for the eventual identification of the endogenous
modulator.
The state of channel modulation does not affect other functional properties of the channel. In both rods and cones, the interaction and permeation of mono- and divalent cations with the channel are unaffected by the state of modulation, as is the voltage dependence of cGMP binding. Since these functional properties likely reflect the structure of the "pore" region of the channel, the interaction between the endogenous modulator and the channel probably occurs in a structural domain distant from the pore.
The interaction between calmodulin, Ca++, and the
target protein is complex. The binding affinity of the
three elements for each other changes depending on
the identity of the target protein. For example, calmodulin in solution binds four Ca++ ions with affinities in
the micromolar range, but in the presence of a target
protein the affinity for Ca++ can be elevated by several
orders of magnitude (reviewed in Klee, 1988; Gnegy,
1995
). Moreover, the Ca++ dependence of a calmodulin-mediated effect on a given target protein changes
with the mole ratio of calmodulin to target protein (Bauer, 1996
). Therefore, the Ca++ sensitivity of the
modulation by calmodulin observed in membrane patches
of rods or cones cannot be assumed to be the same as
in the intact cell unless the mole ratio of channel to
calmodulin were the same. The differences in Ca++ dependence of K1/2 modulation in rod and cone channels
in the presence of calmodulin must reflect differences
in the molecular details of the interaction of calmodulin with the channels.
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
Received for publication 19 June 1997 and accepted in revised form 25 August 1997.
D.H. Hackos is a Howard Hughes Medical Institute Predoctoral Fellow.We thank A. Picones for his patient instruction and continuing interest. We also thank C. Hackos, A. Olson, and T. Rebrik for their valuable comments and discussion.
1. | Alvarez, O., A. Villaroel, and G. Eisenman. 1992. Calculation of ion currents from energy profiles and energy profiles from ion currents in a multibarrier, multisite, multioccupancy channel model. Methods Enzymol 207: 816-854 [Medline]. |
2. | Balasubramanian, S., J.W. Lynch, and P.H. Barry. 1996. Calcium-dependent modulation of the agonist affinity of the mammalian olfactory cyclic nucleotide-gated channel by calmodulin and a novel endogenous factor. J. Membr. Biol. 152: 13-23 [Medline]. |
3. | Bauer, P.J.. 1996. Cyclic GMP-gated channels of bovine rod photoreceptors: affinity, density and stoichiometry of Ca(2+)-calmodulin binding sites. J. Physiol. (Camb.). 494: 675-685 [Abstract]. |
4. |
Baylor, D..
1996.
How photons start vision.
Proc. Natl. Acad. Sci. USA.
93:
560-565
|
5. | Bonigk, W., W. Altenhofen, F. Muller, A. Dose, M. Illing, R.S. Molday, and U.B. Kaupp. 1993. Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels. Neuron. 10: 865-877 [Medline]. |
6. | Chen, T.Y., and K.W. Yau. 1994. Direct modulation by Ca(2+)- calmodulin of cyclic nucleotide-activated channel of rat olfactory receptor neurons. Nature (Lond.). 368: 545-548 [Medline]. |
7. | Cobbs, W.H., A.E. Barkdoll III, and E.N. Pugh Jr.. 1985. Cyclic GMP increases photocurrent and light sensitivity of retinal cones. Nature (Lond.) 317: 64-66 [Medline]. |
8. | Ertel, E.A.. 1990. Excised patches of plasma membrane from vertebrate rod outer segments retain a functional phototransduction enzymatic cascade. Proc. Natl. Acad. Sci. USA. 87: 4226-4230 [Abstract]. |
9. | Fesenko, E.E., S.S. Kolesnikov, and A.L. Lyubarski. 1985. Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segments. Nature (Lond.). 313: 310-313 [Medline]. |
10. | Finn, J.T., M.E. Grunwald, and K.W. Yau. 1996. Cyclic nucleotide-gated ion channels: an extended family with diverse functions. Annu. Rev. Physiol. 58: 395-426 [Medline]. |
11. | Friedberg, F.. 1990. Species comparison of calmodulin sequence. Prot. Seq. Data Anal. 3: 335-337 . |
12. | Frings, S., R. Seifert, M. Godde, and U.B. Kaupp. 1995. Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels. Neuron. 15: 169-179 [Medline]. |
13. | Furman, R.E., and J.C. Tanaka. 1990. Monovalent selectivity of the cyclic guanosine monophosphate-activated ion channel. J. Gen. Physiol. 96: 57-82 [Abstract]. |
14. | Gillespie, P.G., and J.A. Beavo. 1989. Inhibition and stimulation of photoreceptors phosphodiesterase by dipyridamole and M&B 22,948. Mol. Pharmacol. 36: 773-781 [Abstract]. |
15. | Gnegy, M.E.. 1995. Calmodulin: effects of cell stimuli and drugs on cellular activation. Prog. Drug Res. 45: 33-65 [Medline]. |
16. | Gordon, S.E., J. Downing-Park, and A.L. Zimmerman. 1995. Modulation of the cGMP-gated ion channel in frog rods by calmodulin and an endogenous inhibitory factor. J. Physiol. (Camb.). 486: 533-546 [Abstract]. |
17. | Haynes, L.W.. 1992. Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem. J. Gen. Physiol. 100: 783-801 [Abstract]. |
18. | Haynes, L.W.. 1995. Permeation of internal and external monovalent cations through the catfish cone photoreceptor cGMP-gated channel. J. Gen. Physiol. 106: 485-505 [Abstract]. |
19. | Haynes, L.W., and S.C. Stotz. 1997. Modulation of rod, but not cone, cGMP-gated photoreceptor channel by calcium-calmodulin. Vis. Neurosci. 14: 233-239 [Medline]. |
20. | Haynes, L.W., and K.-W. Yau. 1990. Single channel measurement from the cGMP-activated conductance of catfish retinal cones. J. Physiol. (Camb.). 429: 451-481 [Abstract]. |
21. | Horn, R., and J. Patlak. 1980. Single channel currents from excised patches of muscle membrane. Proc. Natl. Acad. Sci. USA. 77: 6930-6934 [Abstract]. |
22. | Hsu, Y.T., and R.S. Molday. 1993. Modulation of the cGMP-gated channel of rod photoreceptor cells by calmodulin Nature (Lond.). 361: 76-79 [Medline]. |
23. |
Hsu, Y.T., and
R.S. Molday.
1994.
Interaction of calmodulin with
the cyclic GMP-gated channel of rod photoreceptor cells. Modulation of activity, affinity purification, and localization.
J. Biol.
Chem.
269:
29765-29770
|
24. | Hurley, J.B.. 1992. Signal transduction enzymes of vertebrate photoreceptors. J. Bioenerg. Biomembr. 24: 219-226 [Medline]. |
25. | Karpen, J.W., A. Zimmerman, L. Stryer, and D.A. Baylor. 1988. Molecular mechanics of the cGMP-activated channel of retinal rods. Cold Spring Harbor Symp. Quant. Biol. 53: 325-332 [Medline]. |
26. | Klee, C.B. 1988. Calmodulin. In Molecular Aspects of Cellular Regulation. P. Cohen and C.B. Klee, editors. Elsevier, Amsterdam, Netherlands. pp. 35-62. |
27. | Klee, C.B., and T.C. Vanaman. 1982. Calmodulin. Adv. Prot. Chem. 35: 213-321 [Medline]. |
28. |
Kohnken, R.E.,
J.G. Chafouleas,
D.M. Eadie,
A.R. Means, and
D.G. Mcconnell.
1981.
Calmodulin in bovine rod outer segments.
J.
Biol. Chem.
256:
12517-12522
|
29. | Korenbrot, J.I.. 1995. Ca2+ flux in retinal rod and cone outer segments: differences in Ca2+ selectivity of the cGMP-gated ion channels and Ca2+ clearance rates. Cell Calcium. 18: 285-300 [Medline]. |
30. | Kosolapov, A.A., and Y. Bobkov. 1996. Modulation of the cGMP- activated conductance of the plasma membrane of photoreceptor cells by calmodulin. Biochem. Mol. Biol. Int. 38: 871-877 [Medline]. |
31. | Kretsinger, R.H.. 1979. The informational role of calcium in the cytosol. Adv. Cyclic Nucleotide Res. 11: 1-26 [Medline]. |
32. | Kurahashi, T., and A. Menini. 1997. Mechanism of odorant adaptation in the olfactory receptor cell. Nature (Lond.). 385: 725-729 [Medline]. |
33. | Lewis, C.A.. 1979. Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction. J. Physiol. (Camb.). 286: 417-445 [Abstract]. |
34. | Liu, M., T.Y. Chen, B. Ahamed, J. Li, and K.W. Yau. 1994. Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. Science (Wash. DC). 266: 1348-1354 [Medline]. |
35. | Martell, A.E., and R.M. Smith. 1974. Critical Stability Constants. Vol. 1. Plenum Press, New York. |
36. | Menini, A.. 1990. Currents carried by monovalent cations through cyclic GMP-activated channels in excised patches from salamander rods. J. Physiol. (Camb.). 424: 167-185 [Abstract]. |
37. |
Miller, D.J., and
G.L. Smith.
1984.
EGTA purity and the buffering
of calcium ions in physiological solutions.
Am. J. Physiol.
246:
C160-C166
|
38. | Miller, J.L., and J.I. Korenbrot. 1994. Differences in calcium homeostasis between retinal rod and cone photoreceptors revealed by the effects of voltage on the cGMP-gated conductance in intact cells. J. Gen. Physiol. 104: 909-940 [Abstract]. |
39. | Miller, J.L., and J.I. Korenbrot. 1993a. In retinal cones, membrane depolarization in darkness activates the cGMP-dependent conductance. A model of Ca homeostasis and the regulation of guanylate cyclase. J. Gen. Physiol. 101: 933-960 [Abstract]. |
40. | Miller, J.L., and J.I. Korenbrot. 1993b. Phototransduction and adaptation in rods, single cones, and twin cones of the striped bass retina: a comparative study. Vis. Neurosci 10: 653-667 [Medline]. |
41. | Molday, R.S.. 1996. Calmodulin regulation of cyclic-nucleotide-gated channels. Curr. Opin. Neurobiol. 6: 445-452 [Medline]. |
42. | Nakamura, T., and G.H. Gold. 1987. A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature (Lond.). 325: 442-444 [Medline]. |
43. | Nakatani, K., Y. Koutalos, and K.-W. Yau. 1995. Ca2+ modulation of the cGMP-gated channel of bullfrog retinal rod photoreceptor. J. Physiol. (Camb.). 484: 69-76 [Abstract]. |
44. | Picones, A., and J.I. Korenbrot. 1995. Permeability and interaction of Ca2+ with cGMP-gated ion channels differ in retinal rod and cone photoreceptors. Biophys. J. 69: 120-127 [Abstract]. |
45. | Picones, A., and J.I. Korenbrot. 1992. Permeation and interaction of monovalent cations with the cGMP-gated channel of cone photoreceptors. J. Gen. Physiol 100: 647-673 [Abstract]. |
46. | Polans, A., W. Baehr, and K. Palczewski. 1996. Turned on by Ca2+! The physiology and pathology of Ca(2+)-binding proteins in the retina. Trends Neurosci. 19: 547-554 [Medline]. |
47. | Pugh, E.N. Jr., and T.D. Lamb. 1993. Amplification and kinetics of the activation steps in phototransduction. Biochim. Biophys. Acta. 1141: 111-149 [Medline]. |
48. | Rebrik, T., and J.I. Korenbrot. 1997. cGMP-gated conductance in intact outer segments of cone photoreceptors with an electropermeabilized inner segment. Investig. Ophthalmol. Vis. Sci. 38: S722 . (Abstr.) . |
49. | Reed, R.R.. 1992. Signaling pathways in odorant detection. Neuron. 8: 205-209 [Medline]. |
50. | Sagoo, M.S., and L. Lagnado. 1996. The action of cytoplasmic calcium on the cGMP-activated channel in salamander rod photoreceptors. J. Physiol. (Camb.). 497: 309-319 [Abstract]. |
51. |
Scott, S.P.,
R.W. Harrison,
I.T. Weber, and
J.C. Tanaka.
1996.
Predicted ligand interactions of 3![]() ![]() |
52. | Weyand, I., M. Godde, S. Frings, J. Weiner, F. Muller, W. Altenhofen, H. Hatt, and U.B. Kaupp. 1994. Cloning and functional expression of a cyclic-nucleotide-gated channel from mammalian sperm. Nature (Lond.). 368: 859-863 [Medline]. |
53. | Williams, D.A., and F.S. Fay. 1990. Intracellular calibration of the fluorescent calcium indicator Fura-2. Cell Calcium. 11: 75-83 [Medline]. |
54. | Yau, K.W., and D.A. Baylor. 1989. Cyclic GMP-activated conductance of retinal photoreceptor cells. Annu. Rev. Neurosci. 12: 289-327 [Medline]. |
55. | Yau, K.-W., and T.-Y. Chen. 1995. Cyclic nucleotide-gated channels. In Ligand- and Voltage-gated Channels. R. Alan North, Editor. CRC Press, Boca Raton, FL. pg. 307-337. |
56. | Zagotta, W.N., and S.A. Siegelbaum. 1996. Structure and function of cyclic nucleotide-gated channels. Annu. Rev. Neurosci. 19: 235-263 [Medline]. |
57. | Zimmerman, A.L., and D.A. Baylor. 1992. Cation interactions within the cyclic GMP-activated channel of retinal rods from the tiger salamander. J. Physiol. (Camb.). 449: 759-783 [Abstract]. |
58. | Zimmerman, A.L., G. Yamanaka, F. Eckstein, D.A. Baylor, and L. Stryer. 1985. Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments. Proc. Natl. Acad. Sci. USA. 82: 8813-8817 [Abstract]. |