From the Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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Activation of cyclic nucleotide-gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide-binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.
Key words: electrophysiology; ion channel gating; kinetics; cyclic GMP; open probability ![]() |
INTRODUCTION |
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Cyclic nucleotide-gated (CNG)1 ion channels of retinal
rod photoreceptors are exquisitely sensitive molecular
detectors of the cyclic nucleotide concentration. The
binding of cyclic nucleotide triggers an allosteric conformational change in the channel protein that opens
the channel pore. In the preceding paper (Sunderman and Zagotta, 1999), we showed that interactions between the purine ring of the cyclic nucleotide and the
binding domain are partially formed at the time of the
transition state for the allosteric transition. These interactions serve to reduce the transition-state energy and
stabilize the activated conformation of the channel relative to the closed state. In this paper, we extend our
analysis of the kinetics of the allosteric transition of bovine rod (BROD) CNG channels by determining the effects on the allosteric transition of mutations at position 604 in the binding domain and of substitution of
the olfactory amino terminal region. These experiments provide insight into the sequence of molecular
events that occur during the conformational change involved in channel activation.
CNG channels contain, in their intracellular carboxyl
terminal region, a domain with significant sequence
similarity to the cyclic nucleotide-binding domain of a
number of other cyclic nucleotide-binding proteins,
including cGMP- and cAMP-dependent protein kinases
and Escherichia coli catabolite gene activator protein
(CAP) (Kaupp et al., 1989). CAP is a cAMP-activated
transcription factor whose structure, while bound to
cAMP, has been determined by x-ray crystallography to
2.5 Å resolution (McKay and Steitz, 1981
; Weber and
Steitz, 1987
). The structure of the cyclic nucleotide-
binding site of CAP consists of eight
strands that
form a
roll structure, followed by two
helices, designated the B helix and C helix. Each cAMP molecule
binds in the anti configuration with the ribose and cyclic phosphate binding to the pocket formed by the
roll and with the N6 hydrogen of adenine hydrogen
bonding with a threonine at position T127 and a serine
at position S128 on the opposite subunit (Weber and
Steitz, 1987
).
Rod CNG channels differ markedly in their apparent
affinities for cGMP, cIMP, and cAMP (Varnum et al.,
1995). To account for the selectivity for cGMP over
cAMP in the rod CNG channel, Varnum et al. (1995)
proposed that the cyclic nucleotides bind in the anti
configuration (Fig. 1). For the case of cGMP, a pair of
hydrogen bonds could then form between the N1 and
N2 hydrogens of the guanine ring of cGMP and the aspartate residue at position 604 in the binding site (Varnum et al., 1995
). Cyclic AMP, which has an unshared pair of electrons at the N1 position, would form an unfavorable electrostatic interaction with the aspartate at
position 604 and thus bind with lower affinity. Alternatively, it has also been proposed for the cGMP-dependent protein kinases and for the cGMP-gated channels
that the cyclic nucleotides bind in the syn configuration, with the N2 hydrogen of guanine hydrogen bonded to a
threonine residue in the
roll found primarily in cGMP-selective proteins (Altenhofen et al., 1991
; Shabb et al.,
1991
; Kumar and Weber, 1992
).
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The amino terminal region of CNG channels has
been shown to affect the free energy of the allosteric
transition (Chen and Yau, 1994; Goulding et al., 1994
;
Gordon and Zagotta, 1995b
). In particular, the amino
terminal region of olfactory CNG channels has been
shown to promote the allosteric transition. This effect of the olfactory amino terminal region is transferable
to rod channels, as chimeric rod channels with the olfactory amino terminal region open more favorably,
and, conversely, chimeric olfactory channels with the
rod amino terminal region exhibit much less favorable
opening (Goulding et al., 1994
; Gordon and Zagotta, 1995b
). Using in vitro protein interaction assays,
specific interactions have been observed between the
amino and carboxyl terminal regions of the olfactory
CNG channel and between the olfactory amino terminal region and the rod carboxyl terminal region (Varnum and Zagotta, 1997
). Similar results were seen with
the rod amino terminal region (Gordon et al. 1997
).
These results indicate that the amino and carboxyl terminal regions of CNG channels bind with high affinity. Thus, conformational changes in the cyclic nucleotide-
binding domain in the carboxyl terminal region could
be transduced to the amino terminal region because of
its close proximity and high affinity interaction. In addition, these results provide a molecular mechanism
for how the gating of olfactory CNG channels can be modulated by Ca2+-calmodulin (Chen and Yau, 1994
; Liu
et al., 1994
; Varnum and Zagotta, 1997
).
In this paper, we analyze the kinetic behavior of single BROD CNG channels in which mutations at amino acid position 604 in the binding domain have been introduced and/or the rat olfactory amino terminal region has been substituted for the BROD amino terminal region. These experiments test the hypothesis that a pair of hydrogen bonds forms between cGMP and D604 during the allosteric transition and probe the mechanism by which the amino terminal region affects the energetics of the allosteric transition. Using a hidden Markov model approach, we analyzed the single-channel records to determine the underlying rate constants. We used thermodynamic mutant cycle formalism to determine the coupling energies for these interactions. By comparing the free energies of the transition state for the allosteric transition relative to the closed and open state energies across mutants and cyclic nucleotides, we postulate a sequence for the molecular events that occur during the allosteric transition.
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MATERIALS AND METHODS |
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Oocyte preparation, cRNA transcription, and expression were
carried out as described previously (Zagotta et al., 1989). Site-specific mutations were generated using oligonucleotide-
directed mutagenesis and PCR and were confirmed by sequencing
as described previously (Gordon and Zagotta, 1995a
). Patch-clamp experiments and analysis of data were carried out as described in the preceding paper (Sunderman and Zagotta, 1999
).
In brief, patch-clamp experiments were performed in the inside-out conformation using an Axopatch 200B amplifier (Axon Instruments). Currents were low-pass filtered at 5 kHz (eight-pole Bessel) and sampled at 25 kHz. Recordings were made at 20-22°C. Initial pipette resistances were 5-20 M
. Intracellular and extracellular solutions contained 130 mM NaCl, 3 mM HEPES, and
0.2 mM EDTA, pH 7.2. For the experiments with Ni2+, 1 µM Ni2+
was substituted for the EDTA. 500 µM niflumic acid was included in the patch pipette to reduce endogenous calcium-activated
chloride currents. Intracellular solutions containing cyclic nucleotides and/or 1 µM Ni2+ were changed using a DAD-12 Superfusion System (Adams and List Associates Ltd.) controlled by a
MRI MB-8000 PC and modified such that each solution had a
separate exit port. All reagents were obtained from Sigma Chemical Co.
Fractional activations (I/Imax) for a particular cyclic nucleotide
were calculated by dividing the current (I) in the presence of the
cyclic nucleotide by the maximum current obtained in the presence of 1 µM Ni2+ plus the best agonist for that channel (Imax). The
fractional activation was used to estimate the free energy change of
the allosteric transition by assuming that the equilibrium constant (L) for this transition is given by: I/Imax = L/(L + 1). Thus the standard free energy for the allosteric transition is G0
RT in L,
where R is the universal gas constant and T is the temperature.
Transition-State Theory
Conversions of rate constants to transition state energies were
made according to Eyring rate theory (Eyring, 1935), which assumes that there is a quasi-equilibrium between the transition state and the ground state and that the rate of break down to product of the high energy intermediate depends on the vibrational energy of a covalent bond at room temperature. While
originally proposed for chemical reactions, this theory has been
applied to conformational changes in proteins and is useful because it provides an estimate of the transition state energies
(Creighton, 1993
). Thus
G
= RT ln(kBT/kobsh) = (17.3
1.35 log kobs) kcal/mol at 22°C, where
G
is the transition state energy, R is the universal gas constant, T is the temperature, kB is
the Boltzmann constant, kobs is the observed transition rate constant, and h is Planck's constant.
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RESULTS |
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To investigate the molecular interactions underlying
the allosteric transition, we recorded macroscopic and
single-channel currents from 10 BROD CNG channel
constructs in which mutations were introduced at position 604 in the binding domain and/or the rat olfactory amino terminal region was substituted for the
BROD amino terminal region (CHM15). The mutations at D604 included D604E (present in the mammalian olfactory subunit), D604Q (present in the fish olfactory
subunit), D604N (present in the rod
subunit), and D604M (present in the olfactory
subunit).
These constructs were expressed as homomultimers in
Xenopus laevis oocytes, and macroscopic currents from
inside-out patches at saturating concentrations of
cGMP, cIMP, and cAMP are shown in Figs. 2-4, respectively.
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Control of Fractional Activation by Amino Acid at Position 604
In Fig. 2 are shown representative current families elicited by voltage steps from 0 mV to between 80 and
+80 mV in the presence of 16 mM cGMP, a saturating
concentration for each of the 10 constructs (Varnum
et al., 1995
). The currents in the absence of cyclic nucleotide were subtracted from each trace. To compare
the currents across experiments, each current family
was normalized to the maximum current obtained at
+80 mV in the presence of 1 µM Ni2+ and the cyclic
nucleotide that best activated the channel. As can be
seen in Fig. 2 A, the fractional activation for BROD with cGMP was greatest for D604, averaging 0.96 ± 0.01 (mean ± SEM, n = 6). The fractional activation was
slightly less (0.81 ± 0.05, n = 4) for the conservative
D604E mutation. When the amino acid at position 604 was mutated to a polar uncharged residue (D604Q or
D604N), there was a dramatic reduction in the fractional activation (D604Q, 0.12 ± 0.03, n = 4; D605N,
0.08 ± 0.02, n = 4). The fractional activation further
decreased to 0.03 ± 0.01 (n = 3) in D604M. Thus, the
fractional activation decreased as the amino acid at position 604 became progressively less polar. Since saturating concentrations of cyclic nucleotide were used in
all of these experiments, a reduction in fractional activation indicates that the allosteric conformational
change for fully liganded channels was less favorable
for the mutant channels than for the wild-type channel.
The dramatic reduction in fractional activation from 0.96 for D604 down to 0.03 for D604M is a testament to
the importance of D604 for the allosteric transition.
This result is consistent with what has been previously
reported and has been proposed to result from hydrogen bonding between the carbonyl group of D604 and
the guanine ring of cGMP (Varnum et al., 1995
).
For comparison, the same mutations at position 604 were also studied in the CHM15 background. CHM15
is identical to the BROD construct except that the rat
olfactory amino terminal region has been substituted
for the BROD amino terminal region (Gordon and
Zagotta, 1995b). The olfactory amino terminal region
produces a more favorable free energy change for the
allosteric transition of chimeric channels (Goulding et al.,
1994
; Gordon and Zagotta, 1995b
), making the effects
of mutations that dramatically decrease the ability of cyclic nucleotides to promote the allosteric transition easier to characterize at both the macroscopic and single-channel levels. In Fig. 2 B are illustrated the currents in
the CHM15 background for the D604 mutants in the
presence of cGMP. Again as the amino acid at position
604 was made progressively less polar, the fractional activation decreased. The fractional activities were 0.96 ± 0.02 (n = 4) for CHM15-D604, 0.98 ± 0.01 (n = 3) for CHM15-D604E, 0.90 ± 0.01 (n = 3) for CHM15-D604Q, 0.82 ± 0.02 (n = 5) for CHM15-D604N, and
0.52 ± 0.07 (n = 3) for CHM15-D604M. We interpret
these results to indicate that, compared with the BROD
background, the trend across constructs was similar, although the differences in fractional activation were
smaller. Despite smaller effects on the fractional activations, the effects of the D604 mutations on the free energy of the allosteric transition is the same in both the
BROD and CHM15 backgrounds (see Table I).
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Cyclic IMP is similar in chemical structure to cGMP except that the inosine moiety lacks a 2-amino group. Therefore, different abilities of cGMP and cIMP to promote the allosteric transition should reflect the contribution of interactions of the cyclic nucleotide-binding domain of the channel with the guanine 2-amino group of cGMP to the allosteric transition. We recorded macroscopic currents from the 10 constructs activated by cIMP, and these currents are illustrated in Fig. 3, A and B, for the BROD and CHM15 backgrounds, respectively. As can be seen in this figure, the fractional activation was less for the D604Q, D604N, and D604M mutants than for D604 and D604E in both the BROD and CHM15 backgrounds. In BROD D604, the fractional activation by cIMP was only 0.60 ± 0.02 (n = 6) and, for BROD D604E, the fractional activation was slightly lower (0.41 ± 0.05, n = 4). For D604Q and D604N, the fractional activations were 0.05 ± 0.01 (n = 4) and 0.08 ± 0.02 (n = 4), respectively. For D604M, the fractional activation was 0.06 ± 0.004 (n = 4). For the CHM15 constructs, the fractional activations were 0.97 ± 0.01 (n = 4) for D604, 0.98 ± 0.01 (n = 3) for D604E, 0.73 ± 0.05 (n = 5) for D604Q, 0.84 ± 0.03 (n = 5) for D604N, and 0.71 ± 0.03 (n = 3) for D604M. Thus, mutating D604 to a polar uncharged residue (Q or N) or nonpolar uncharged residue (M) decreased the fractional activation. Compared with the effects for cGMP, the differences in fractional activation were smaller, suggesting that cIMP is less sensitive to the identity of the amino acid at position 604. This finding likely reflects cIMP's lesser potential for hydrogen bonding interactions than cGMP's. Thus, the energetic effects of mutations at position 604 would be expected to be smaller for cIMP than for cGMP.
Like cIMP, cAMP lacks a 2-amino group, but it has
two other differences in the purine ring. cAMP has an
amino group instead of a carbonyl group at the 6-position, and it has an unshared pair of electrons instead
of a hydrogen at the 1-position. In Fig. 4 are illustrated
currents activated by 16 mM cAMP. Here the pattern
across the constructs was quite different from the pattern that was observed with cGMP and cIMP. For
D604, the fractional activation was only 0.012 ± 0.002 (n = 6) or almost two orders of magnitude less than for
cGMP on the same construct. For D604E, the activation
was almost twice as large but still small: 0.02 ± 0.01 (n = 3). For D604Q and D604N, the activation increased to
0.05 ± 0.01 (n = 4) and 0.05 ± 0.02 (n = 4), respectively. For D604M, the fractional activation was significantly larger: 0.18 ± 0.01 (n = 3). For the CHM15 constructs, the fractional activation was 0.19 ± 0.02 (n = 4)
and 0.53 ± 0.03 (n = 3) for D604 and D604E. For
D604Q and D604N, the fractional activations were 0.74 ± 0.04 (n = 5) and 0.69 ± 0.03 (n = 6), respectively. For
D604M, the activation was 0.90 ± 0.03 (n = 3). Thus, as
the amino acid at position 604 was made progressively
less polar, cAMP became a better agonist. The molecular interpretation of this result is that the reduction in
the polarity of the residue at position 604 reduces the
unfavorable interaction that would be expected to occur between the unshared pair of electrons at the N1
position of cAMP and a polar residue at 604. The slight improvements in the factional activations by cAMP of
D604E over D604 may be related to differences in the
local pKa's of aspartate and glutamate. Specifically,
Gordon et al. (1996) showed that the fractional activation by cAMP on D604 improves as the pH is decreased.
The cyclic nucleotide-specific pH effect disappeared when D604N was introduced, suggesting that cAMP is
sensitive to the protonation state of D604. The pH dependence was proposed to result from neutralization of
a negative electrostatic interaction between the negative charge on 604N and the unshared pair of electrons at the 1-position of the purine ring of cAMP. At pH 7.2, D604 would be unprotonated, but it is possible that
D604E is partially protonated at pH 7.2, thereby improving the fractional activation by cAMP by removing
some of the negative charge on the carboxylic acid.
Thermodynamic Mutant Cycle Analysis
To summarize the results from the macroscopic experiments, we converted the fractional activations to free
energies for the allosteric transition (see MATERIALS
AND METHODS) and used these energies to construct
thermodynamic mutant cycles. Thermodynamic mutant cycles provide a way of separating out indirect effects of mutations on the machinery of the allosteric
transition from direct interactions of the ligand with
the ligand-binding domain. Thermodynamic mutant
cycles have been applied to interactions between the
amino acids within proteins (Carter et al., 1984; Horovitz
et al., 1990
; Serrano et al., 1990
) and between a toxin and a voltage-dependent K+ channel (Hidalgo and
MacKinnon, 1995
). We wanted to determine if the effects of mutations at 604 reflect direct interactions between D604 and the places on the purine rings where
the cyclic nucleotides differ. An example of the use of
thermodynamic mutant cycles is shown in Fig. 5. In Fig.
5 A, we tested for direct interaction of the amino terminal region with the purine ring of the cyclic nucleotide.
The horizontal arrows reflect the effect of the amino terminal substitution on the free energy change of the
allosteric transition, while the vertical arrows reflect the
effect of changing cyclic nucleotide on the free energy
change of the allosteric transition for the particular
channel constructs. We would expect that the effect of
the mutation might have two components: (a) a nonspecific component relating to indirect effects on the
allosteric transition machinery and (b) a specific component due to direct effects on the interaction of the
ligand with D604. As can be seen in Fig. 5 A, the
Gs
for both of the horizontal arrows are negative, indicating that the olfactory amino terminal region makes the
allosteric transition more favorable for both cGMP and cAMP. The difference between the values of the two
Gs should subtract out the nonspecific effect while
leaving the direct effects. This difference is referred to
as the coupling energy. Since CNG channels have four
subunits (Liu et al., 1996
; Varnum and Zagotta, 1996
),
the coupling energies reflect the sum of the direct interactions occurring in each subunit. For this cycle, the
coupling energy was only 0.49 kcal/mol, indicating that
the effect of substituting the amino terminal region is
relatively cyclic nucleotide independent. This result
was an expected finding since it has been previously shown that the effect of the olfactory amino terminal
region is cyclic nucleotide independent (Liu et al.,
1994
; Goulding et al., 1994
; Gordon and Zagotta,
1995b
). Thus, the differences in the way cAMP and
cGMP interact with the channel because of the differences in their purine ring structures are preserved on
substituting the olfactory amino terminal region. That
the effect of the olfactory amino terminal region was
cyclic nucleotide independent was also reflected in the
macroscopic traces; the trends across the D604 mutants
were similar in both the CHM15 and BROD backgrounds.
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Illustrated in Fig. 5 B is a thermodynamic mutant cycle
analysis of the interaction between D604 and the cyclic
nucleotide. Here the D604M mutation makes cGMP a
worse agonist (G = 4.09 kcal/mol), but makes cAMP
a better agonist (
G =
1.79 kcal/mol). Equivalently,
the cyclic nucleotide selectivity was inverted by the
D604M mutation, as evidenced by the negative value
for
G (
G =
1.00 kcal/mol) between cGMP and
cAMP on D604M and positive value for
G (
G = 4.88 kcal/mol) between cGMP and cAMP on D604. Here, the coupling energy was large and negative (coupling energy =
5.88 kcal/mol), indicating a high degree of interaction between the amino acid at position
604 and the purine ring of the cyclic nucleotide.
Summarized in Table I are the coupling energies for
each of the possible thermodynamic mutant cycle comparisons for the 10 different mutants with the three different cyclic nucleotides. All energies of magnitude
2.5 kcal/mol are double underlined, and all energies
of magnitude between 2 and 2.5 kcal/mol are single underlined. As seen along the diagonal, the coupling
energies of changes in the amino terminal region with
changes in cyclic nucleotide were generally small. We
interpret this to mean that the effect of the olfactory
amino terminal region was cyclic nucleotide independent. Note that the coupling energies between CHM15
and BROD were slightly larger than for the other comparisons, probably because the fractional activations of
cGMP and cIMP on CHM15 were so large that the
method of using Ni2+ potentiation to measure
G0
loses resolution. In addition, all of the cells comparing
the BROD-D604M and CHM15-D604M to the BROD,
CHM15, BROD-D604E, and CHM15-D604E between
cIMP and cAMP and between cGMP and cAMP are underlined. This result strongly indicates that cAMP is
sensing the amino acid at position 604 quite differently
from cIMP and cGMP. This result gives good support
for the hypothesis proposed by Varnum et al. (1995)
that an acidic residue at position 604 in BROD channels is critical for ligand discrimination and interacts directly with the purine ring of the cyclic nucleotide
(Fig. 1). This conclusion is also supported by the fact
that the majority of the cells for the more conservative
D604Q and D604N vs. D604 and D604E in the BROD
and CHM15 backgrounds were also >2 kcal/mol for
cIMP vs. cAMP and cAMP vs. cGMP.
For the cGMP vs. cIMP comparisons, the coupling
energies were smaller in magnitude, as expected since
the chemical differences between cGMP and cIMP are
smaller relative to their differences with cAMP. As can
be seen in Table I, there was a 2 kcal/mol coupling
energy in the BROD vs. BROD-D604N, CHM15-D604N,
BROD-D604M, and CHM15-D604M. Since the only difference between cGMP and cIMP is at the 2-positions of
their purine rings, this finding supports the hypothesis
of Varnum et al. (1995)
that the guanine 2-amino group
of cGMP interacts with D604 (Fig. 1). For CHM15, the
same analysis did not reveal large coupling energies
with the same four constructs vs. CHM15. However, we
do not feel that this negative result indicates a lack of
interaction. Rather, it is probable that we were not able
to pick up a differential interaction because the
CHM15 construct had such a favorable
G0 for the allosteric transition so that the currents were nearly maximal without Ni2+ in the presence of cGMP and cIMP.
For BROD-D604E and CHM15-D604E, the coupling
energies with BROD-D604M and CHM15-D604M
ranged from
0.92 to
1.46 kcal/mol. These values
are smaller than the values of
2.21 and
2.30 kcal/
mol between BROD and BROD-D604M and CHM15-D604M, possibly reflecting weaker interactions because
of the larger side chain of glutamate over aspartate.
Thus, overall, we conclude that the comparisons between the D604 and D604E constructs and the D604M
constructs provide support for the hypothesis of Varnum et al. (1995)
that cIMP forms one hydrogen bond
with D604 while cGMP forms two. The coupling energies we measured are within the range of energies expected for such an interaction (Fersht et al., 1985
).
Effects of Position 604 Mutations Are on Gating, not Single-Channel Amplitude
To probe the effects on the opening and closing rate
constants, not just the overall G0, we obtained single-channel recordings at +80 mV for each of the 10 constructs in the presence of saturating concentrations of
cGMP, cIMP, and cAMP. From each recording, we selected regions where a single channel was active, and
we omitted from our analysis quiescent periods 200 ms
or longer in duration (Sunderman and Zagotta, 1999
).
Representative 200-ms portions of our recordings are illustrated in Figs. 6, 8, and 10 for cGMP, cIMP, and
cAMP, respectively. The amplitude histograms for the
recordings shown in Figs. 6, 8, and 10 are shown in
Figs. 7, 9, and 11.
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Fig. 6 shows representative single-channel traces for the 10 constructs activated by 16 mM cGMP. As 16 mM cGMP is a saturating concentration, the kinetics that we observe do not reflect the rate constants of binding or unbinding of cGMP. Rather, they reflect transitions after the full complement of ligands has bound to the channel. For D604, the channel was highly activated in the BROD construct, but more so for CHM15. In both cases, the single-channel conductance was the same. We compare the open probabilities and single-channel current level in the amplitude distributions in Fig. 7, which shows BROD and CHM15 distributions side-by-side. As expected given the slightly lower fractional activation of D604E compared with D604 in macroscopic experiments, the open probability of D604E was slightly lower than for D604. For BROD-D604Q and BROD-D604N, the open probability decreased substantially. For BROD-D604M, the open probability was very low but increased to ~0.5 in CHM15-D604M. In each case, the single-channel conductance appears to be unaffected by mutations at position 604 or the presence of the olfactory amino terminal region, and openings appear to be to only the full amplitude level, not to subconductance states. Thus, the large differences in fractional activation determined from the macroscopic current experiments are due entirely to differences in the open probabilities produced by D604 mutations and the olfactory amino terminal region. In particular, the open probabilities in cGMP decreased as D604 became less polar and increased with the addition of the olfactory amino terminal region.
For cIMP activation of the 10 constructs, representative traces and amplitude histograms are shown in Figs. 8 and 9, respectively. Here, the trend across the constructs was quite similar to the trend for cGMP, although there were a few differences. With cIMP, the open probability was only 74% in D604. Like for cGMP, the open probability decreased as the amino acid at position 604 became less polar and increased with the addition of the olfactory amino terminal region. Interestingly, openings in BROD-D604M were slightly more numerous and longer-lived than for cGMP.
For cAMP activation across the 10 constructs, representative traces and amplitude histograms are shown in Figs. 10 and 11. As can be seen here, the open probability was low when a polar residue was present at position 604. For BROD-D604M, the openings were significantly longer in duration. The same trends were observed in the CHM15 constructs.
Hidden Markov Model Analysis of Single-Channel Patch-Clamp Recordings
We analyzed the single-channel kinetics for each of the
10 constructs using a hidden Markov modeling (HMM)
approach. The HMM approach, described in detail in
the accompanying paper (Sunderman and Zagotta,
1999), directly estimates the rate constants for a given
specified kinetic scheme and uses iterative techniques to converge on the maximum likelihood set of rate
constants. The HMM approach provides a maximum
likelihood value that allows the number of closed and
open states required to explain the data to be determined. In the previous paper, we found that a simple two-state C
O scheme is not sufficient to explain the
kinetics at saturating ligand concentrations. Rather, an
additional closed state was required. Adding this additional closed state outside the activation pathway (as in
C0
O1
C2) or within the activation pathway (as in
C0'
C1'
O2') significantly improved the description
of the kinetics at saturating ligand concentrations.
Since the likelihoods for the C0
O1
C2 and C0'
C1'
O2' linear schemes are identical, we could not
discriminate between these two schemes. However, we
prefer the C0
O1
C2 scheme because there was cyclic nucleotide dependence in only the first transition
of the C0
O1
C2 scheme. Also, the C0
O1
C2
scheme has the simple physical interpretation of the
first transition being the allosteric transition and the second transition being to a flicker closed state out of
the activation pathway. While the underlying mechanism is undoubtedly more complex than two closed
and one open states, this mechanism adequately describes our data and provides a physical interpretation
of the results. The qualitative conclusions concerning
the effects of mutations on the stability of the open
state and transition state should still be valid for a C0'
C1'
O2' scheme and more complex models. These
conclusions are based on effects of these mutations on
the open and closed durations and are not dependent
on any particular model.
For the analysis of the 10 constructs described in this
paper, we determined the rate constants for the C0 O1
C2 scheme. Shown in Fig. 12 is a summary of the
rate constants from multiple patches for the C0
O1
step for all 10 constructs and for all three cyclic nucleotides. The opening rate k01 became progressively slower for cGMP as the amino acid at position 604 became less polar (Fig. 12 A). This effect was observed in
both the BROD and CHM15 constructs. For cIMP, the
slowing of the k01 rate was smaller but in the same direction. For cAMP, the k01 rate constant was nearly independent of construct, perhaps increasing slightly. In
Fig. 12 B, we see that the effects of the D604 mutations
on the k10 rate constant are larger than for the k01 rate
constant. For cGMP, there was a progressive speeding
up of the closing rate (k10) constant as the amino acid
at position 604 became less polar. For cIMP, the trend
across constructs was generally the same but smaller in
magnitude. For cAMP, the k10 rate constant was relatively independent of the amino acid at position 604, thus indicating that the interactions of cAMP with
D604 are unimportant for determining the closing rate
constant.
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For the CHM15 constructs, the effect on the k10 rate constant was remarkable. For each mutation at position 604 in the BROD construct, the corresponding mutation in the CHM15 construct was ~20-100-fold slower. This result contrasts with the result for the opening rate constant, which appeared to be relatively independent of the presence or absence of the olfactory amino terminal region. The large effect on the closing rate constant indicates that the positive interactions incurred with the substitution of the olfactory amino terminal region are a major determinant of the closing rate constant.
Shown in Fig. 13 are the rate constants for the second
transition O1 C2 across constructs and for the three
cyclic nucleotides. As can be seen in this figure, the reopening rate constant k21 was generally independent of
cyclic nucleotide and construct and was fast (~5,000/s).
The closing rate k12 was also generally independent of
cyclic nucleotide and construct but was much slower
(~100/s). Thus, the equilibrium for the O1
C2 step
is strongly toward the open state, and sojourns in the C2
state were short in duration, not unlike what would be expected for flickery open-channel block. More importantly, the effects of the D604 mutations and the olfactory amino terminal region are selective for the first transition
of C0
O1
C2, as was previously shown for the effects
of cyclic nucleotides and Ni2+ (Sunderman and Zagotta,
1999
). The observation that each of these modifications
is affecting the same step provides further support for the
hypothesis that the first transition of the C0
O1
C2
scheme is the allosteric transition, and the second transition is not involved in the allosteric transition.
|
Correspondence between Macroscopic and Single-Channel Current Recordings
A comparison between the values for G0 calculated as
RT ln(k01/k10) from the single-channel experiments,
relative to the values for
G0 =
RT ln L, where L is the
equilibrium constant for the allosteric transition and was
determined using Ni2+ potentiation of macroscopic experiments, is shown in Fig. 14 (see MATERIALS AND METHODS). Overall, the correspondence between the values is
good. It may be noted that for BROD D604Q, D604N,
and D604M, the median single-channel estimate for
G0
was slightly larger than the macroscopic estimate in each
case. The probable explanation for this observation is
that currents obtained with Ni2+ in macroscopic experiments slightly underestimate the maximum current. This
underestimation is occurring because the
G0 for the
transition is so unfavorable for each of these constructs that, even with Ni2+, the currents do not approximate the
theoretical maximum current that would be obtained if
the fractional activation were 1. Note that this slight difference could not be explained by error in the determination of the number of channels in a single-channel patch, as the effect of having too many channels would
be to increase the opening rate constant, thus making
the
G0 more favorable, not less. This figure illustrates
that there was generally good correspondence between
the values obtained for the macroscopic and single-channel experiments. This result supports the use of Ni2+ for
the estimation of
G0 from macroscopic experiments
and provides additional evidence that the C0
O1 transition represents the transition modulated by Ni2+.
|
Effects of Allosteric Modulation on the Free Energy Profiles for the Allosteric Transition
Using Eyring rate theory (Eyring, 1935), we converted
the median rate constants we obtained from our HMM
computations to activation energies (see MATERIALS
AND METHODS). Eyring rate theory assumes that there is
a quasi-equilibrium between a high energy transition state and the ground state, and that the rate of break
down to product of the high energy intermediate depends on the vibrational energy of a covalent bond at
room temperature. Other theories have been proposed
to explain the slow rate of protein conformational changes (Jackson, 1993
); however, Eyring rate theory is
generally accepted and provides a unique estimate of
the transition state energies (Creighton, 1993
). While
the absolute activation energy (
G
) is dependent on estimates of a preexponential factor, the changes in activation energy (
G
) are less dependent on this estimate.
Fig. 15 A illustrates the energetics for the allosteric
transition for the three cyclic nucleotides. In this figure, the energy profiles were aligned at 0 kcal/mol
when the channels were in the closed state (C0). As can
be seen in this figure, the overall G0 for the allosteric
transition is between
2 and 3 kcal/mol for cGMP and
cAMP, respectively. Since these cyclic nucleotides differ in only the most distal portion of their purine ring, we
conclude that the cyclic nucleotide-binding domain interacts with the purine ring of the cyclic nucleotides
differently during the allosteric transition. The energetics for these favorable cyclic nucleotide-binding domain interactions must therefore be determinants of
the stability of the open state. Since the energy of the
transition state (
G
) also differs among the cyclic nucleotides, we conclude that these interactions are partially formed at the time of the transition state and
serve to reduce the energetic barrier for activation.
|
The effects of Ni2+ presented in the previous paper
(Sunderman and Zagotta, 1999) on the transition-state
energies are diagrammed in Fig. 15 B. This figure illustrates that, like the cyclic nucleotides, Ni2+ also affects
the transition state energy and stability of the open
state relative to the closed state. For both cIMP and
cAMP, the presence of Ni2+ stabilizes the open state
and has a small effect on the energy of the transition
state. This finding suggests that the movement of the
H420 residues on each of the subunits of the channel
into the Ni2+ coordinating position is associated with
channel opening. These interactions between Ni2+ and
the channel are partially formed at the time of the transition state for the allosteric transition.
Fig. 15 C shows the energetics for activation of BROD and BROD-D604M channels by cGMP. As can be seen in this figure, D604M channels activated by cGMP have an allosteric transition energetically very similar to BROD channels activated by cAMP, both with regard to the standard free energy of the transition and the activation energy. This is a testament to the idea that the purine ring of cGMP interacts directly with D604. Disrupting this interaction with either cAMP or D604 mutations has a very similar effect. Fig. 15 D shows the energetics for activation of BROD and CHM15 channels by cIMP. As can be seen in this figure, the mechanism of action of the olfactory amino terminal region is a stabilization of the open state without affecting the transition state energy. This finding suggests that the stabilizing interactions between the autoexcitatory domain of the olfactory amino terminal region do not form until after the transition state for the allosteric transition. Thus, the interactions of the cyclic nucleotide and the binding site form at the time of the transition state, while the stabilizing effect of the olfactory amino terminal region arises after the transition state.
Fraction of Energetic Effect after the Transition State Reveals the Sequence of Events
To probe the sequence of events underlying the allosteric transition, we calculated the fraction of the energetic effect of the modifications studied that occurs after the allosteric transition and plotted the median values in Fig. 16. These calculations were made by
considering the effect on the k01 and k10 rate constants of switching cyclic nucleotide on BROD-D604 channels, switching from D604 to D604M in either the
BROD or CHM15 backgrounds and for each of the cyclic nucleotides, applying Ni2+ on BROD channels
(Sunderman and Zagotta, 1999), and switching from
BROD-D604 to CHM15-D604 for each of the cyclic nucleotides. As can be seen in Fig. 16, in each case, the
majority of the energetic effect of each modification
occurred on the closing rate constant, meaning after
the transition state for the allosteric conformational change. Intriguingly, the median fraction of the effect
occurring after the transition state was 68% for the interactions of the channel with the portions of the cyclic
nucleotides that differ and 70% for the effect of switching to D604M. The close correspondence between
these two values gives further support for the hypothesis that the purine rings of the cyclic nucleotides and
the amino acid at position 604 interact directly because
it indicates that the D604M mutation and switching to a
different cyclic nucleotide not only affect both rate
constants, but they affect both rate constants commensurately. For Ni2+, there was more variation in the fraction of the energetic effect occurring after the transition state. For the olfactory amino terminal region, the
median value was strongly skewed toward the right, indicating that only a small fraction of the energetic effect of the amino terminal region has occurred by the
time of the transition state. Hence, we would say that
the state-dependent stabilization of the allosteric transition occurs very late in the reaction coordinate. It is
tantalizing to conclude that the interactions between
the purine ring of the cyclic nucleotide and the amino
acid at position 604 occur early in the reaction coordinate. These interactions are followed by the interactions of H420 in the C linker with Ni2+. Finally, the stabilizing interactions with the olfactory amino terminal
region, when present, form late in the reaction coordinate, after the channel has switched to the active conformation.
|
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DISCUSSION |
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In this paper, we have investigated the effects of mutations at position 604 in the binding domain and of the olfactory amino terminal region on the kinetics at saturating concentrations of three different ligands. We have shown that direct interactions between the cyclic nucleotide and the amino acid at position 604 occur during both the opening and closing transitions of the allosteric conformational change. In contrast, the substitution of the olfactory for the rod amino terminal region appears to affect only the closing rate constant, suggesting that the favorable interdomain interactions that form when the auto-excitatory domain is present occur almost entirely after the channel has switched to the open conformation.
A molecular mechanism for the binding of cyclic
nucleotides to CNG channels has been proposed,
based on the CAP structure (Gordon and Zagotta,
1995b; Varnum et al., 1995
). In this mechanism, the cyclic nucleotide-binding site is exposed when the channel is in the closed configuration because the C helix is
rotated away from the
roll. The cyclic nucleotide
then binds to the closed channel primarily through interactions between the
roll and the ribose and phosphate of the cyclic nucleotide. Activation involves a
conformational change in the channel protein. At the level of the binding site, the conformational change is
thought to involve movement of the
roll relative to
the C helix, permitting residues in the C helix, in particular D604, to interact specifically with the purine
ring of the bound cyclic nucleotide. For cGMP, it was
proposed that the negatively charged carboxylic acid
side chain of D604 in the C helix forms a pair of hydrogen bonds with the N1 and N2 hydrogen atoms on the
guanine ring. This type of hydrogen bonding has been
shown to occur in solution (Lancelot and Hélène,
1977
) and in high affinity GTP binding proteins such
as the
subunit of transducin (Noel et al., 1993
), elongation factor EF-Tu (Jurnak, 1985
), and H-Ras (Pai et al.,
1989
). This molecular mechanism is not unique, and
we cannot completely rule out that substituents on the
purine ring do not alter the chemical properties of the
ring, or that mutations at D604 do not have indirect effects on the structure of the cyclic nucleotide-binding site. However, given the large body of data in support of
a direct interaction between D604 and the purine ring,
we have chosen to interpret our results in terms of this mechanism.
Based on the results of this investigation, the kinetic and molecular aspects of this mechanism for the allosteric transition have been refined. In particular, we have determined the rate constants at which the interactions are forming and unforming, and we now know the relative effects on the opening and closing transitions for each of the modifications studied. For the case of cGMP, we conclude that the pair of hydrogen bonds between the guanine ring and the aspartate is partially formed at the time of the transition state for the allosteric transition. Thus, the activation of the channel involves the formation of these two hydrogen bonds, and the closing of the channel involves the dissolution of these hydrogen bonds. This observation indicates that the formation of these hydrogen bonds is intimately involved in the allosteric transition, not an event that occurs before or immediately after it. For cIMP, we conclude that the formation of a hydrogen bond between D604 and the inosine moiety is also intimately involved in the allosteric transition, but the energies are approximately half those of cGMP, indicating a molecular mechanism where only a single hydrogen bond forms per subunit. The open probability increased for activation by cAMP when D604 was mutated to a polar uncharged residue, suggesting that interactions between the adenine ring and D604 destabilize the allosteric transition. Thus, other portions of the cyclic nucleotide and interactions not investigated here may explain the fact that cAMP is an agonist, not an antagonist, of the BROD channel. Alternatively, D604 may interact weakly with the adenine ring and less polar residues may interact more strongly.
Our analysis of the effect of the olfactory amino terminal region on channel activation indicates that the
amino terminal region exerts its auto-excitatory effect
late in the reaction coordinate and that the effects of
the olfactory amino terminal region are cyclic nucleotide-independent. Previously, it has been shown that
the rat olfactory CNG channel amino terminal region promotes channel activation by directly interacting
with the carboxyl terminal gating machinery (Varnum
and Zagotta, 1997). This mechanism was proposed
based on in vitro binding assays and occurred even in
the absence of cyclic nucleotide. The results of this investigation indicate that the affinity of the two terminal
regions is only 2 kcal/mol higher for the open than the
closed state. Since this energy difference is small, the
most likely interpretation is that the two regions interact with high affinity both when the channels are open
and closed and that the 2 kcal/mol energetic stabilization reflects the formation of a few hydrogen bonds per subunit or the removal of a few unfavorable interactions and not the binding or unbinding of the two terminal regions with each transition. Thus, our results refine the molecular mechanism of action by suggesting
that the stabilization exerted by the amino terminal region occurs primarily after the transition state for the
allosteric transition and occurs later in the reaction coordinate than the interactions between the cyclic nucleotides and the amino acid at position 604.
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FOOTNOTES |
---|
Original version received 11 November 1998 and accepted version received 10 March 1999.
We thank Heidi Utsugi and Kevin Black for technical assistance, Fred Sigworth and Lalitha Venkataramanan for helpful discussions, and Richard W. Aldrich and Anita Zimmerman for comments on the manuscript.
This work was supported by the Howard Hughes Medical Institute and by a grant from the National Eye Institute (EY10329) to W.N. Zagotta. W.N. Zagotta is an investigator of the Howard Hughes Medical Institute.
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Abbreviations used in this paper |
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BROD, bovine rod; CAP, catabolite gene activator protein; CNG, cyclic nucleotide-gated; HMM, hidden Markov model.
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