Correspondence to: Anna Menini, Biophysics Sector, International School for Advanced Studies, Via Beirut 2, 34014 Trieste, Italy. Fax:39-040-2240470 E-mail:menini{at}sissa.it.
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Abstract |
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Upon stimulation by odorants, Ca2+ and Na+ enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotidegated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotidegated channels play a similar role. We studied the corresponding glutamate (E340) of the
subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca2+ and Mg2+. E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca2+ and Mg2+ powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca2+ relative to Na+ was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca2+ and Mg2+, and affects channel gating by cAMP more than by cGMP.
Key Words: cyclic guanosine monophosphate, cyclic adenosine monophosphate, olfactory sensory neurons, site-directed mutagenesis
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INTRODUCTION |
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Olfactory transduction is initiated by the binding of odorant molecules to receptor proteins located in the cilia of olfactory sensory neurons. This binding causes the activation of a receptor-coupled G protein, which in turn activates an adenylyl cyclase that synthesizes cAMP. The resultant increase in cAMP concentration leads to the opening of cyclic nucleotidegated (CNG)1 ion channels in the ciliary membrane (
Native CNG channels from olfactory sensory neurons have been supposed to be heterotetrameric complexes with two types of subunits, named differently by different authors. However, a third type of subunit, a short splice variant of the retinal rod ß subunit, has recently been cloned (3 for the principal olfactory
subunit,
4 for the second cloned subunit (this subunit was originally named rOCNC2 by
3 subunit expressed in Xenopus laevis oocytes forms functional channels (
4 and ß1b, are modulatory subunits and do not form functional CNG channels on their own. The
3 subunit has a glutamate residue (E340) that corresponds to E363 of the
subunit of the bovine rod CNG channel. The two subunits
4 and ß1b have an aspartate and a glycine residue, respectively, at the corresponding location.
Each CNG channel subunit is formed by six transmembrane segments, with the pore region located between the fifth and sixth transmembrane segment (for reviews, see
A number of previous studies have reported that extracellular Ca2+ effectively blocks the current carried by monovalent ions through retinal rod, cone, and olfactory CNG channels in a voltage-dependent way (for reviews, see subunit of CNG channels from bovine retinal rods showed that blockage by external Ca2+ and Mg2+ critically depends on E363 (
The aim of the present study was to investigate the role in permeation, gating, and block by Ca2+ and Mg2+ of the residue at position 340 in the 3 subunit of the bovine olfactory CNG channel.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis and Functional Expression
Site-directed mutagenesis was performed following the procedure of
Current Recordings
Current measurements were performed from patches excised from the plasma membrane of oocytes in the inside-out or outside-out configuration.
Macroscopic currents were measured with the Axopatch 1D patch-clamp amplifier (Axon Instruments, Inc.), filtered at 1 kHz, and sampled at 2 or 2.5 kHz using a TL-1 DMA interface board (Axon Instruments, Inc.) with a PC-type computer and pClamp5 software (Axon Instruments, Inc.).
Single-channel recordings, performed 13 d after mRNA injection, allowed the determination of single-channel current and open probability of wild-type and mutant channels. Single-channel traces were recorded filtering at 2 kHz and sampled at 10 kHz. All-points amplitude histograms were constructed from 30 s of data recordings. The single-channel current and the open probability were determined by analyzing amplitude histograms normalized to a total area of 1. Histograms were fitted using the Levenberg-Marquardt least square algorithm by the sum of two or more Gaussian functions with pClamp6 software (Axon Instruments, Inc.).
Ionic Solutions
The ionic composition of the divalent cation-free solution at the extracellular and intracellular sides of the membrane patch was: 110 mM NaCl, no added divalent salts, 2 mM EDTA, and 10 mM HEPES, buffered to pH 7.4 with NaOH or with tetramethylammonium hydroxide. Low Ca2+ or Mg2+ concentrations were buffered by 2 mM nitrilotriacetic acid or by EDTA, respectively. The concentrations of CaCl2 or MgCl2, necessary to give the indicated free Ca2+ or Mg2+ concentrations were calculated using the chelator program of
Channels were activated with various concentrations of cyclic nucleotides, cGMP or cAMP, applied at the intracellular side of an excised patch. Exchange of bath solutions was achieved by positioning the excised patch in front of one of four glass pipes from which test solutions were continuously flowing, as previously described by
CurrentVoltage Relations
Macroscopic currentvoltage relations were measured by applying voltage steps of 150-ms duration from a holding potential of 0 mV in ±20-mV steps. For the measurement of the current blockage by divalent cations, currentvoltage relations were obtained by ramping the voltage command from -80 to +80 mV in 1 s; five individual ramps were averaged in each measurement. I-V relations at the beginning of each experiment were also measured by applying voltage steps of 150-ms duration to investigate whether the patch presented ion accumulation effects, as described by
Outside-out patches were used to investigate the blockage by external Ca2+ and Mg2+. In these experiments, 1 mM cGMP was present in the patch pipette, unless otherwise indicated. Similar results were obtained when channels were activated by cAMP. The leak conductance was assumed not to be voltage dependent, and was usually estimated by perfusing the external side of the patch with a solution containing 73.3 mM CdCl2 or MgCl2 buffered to pH 7.4 with tetramethylammonium hydroxide. The residual conductance measured at negative potentials was taken as the leak conductance and was subtracted from each I-V relation. Further experiments were only performed when the leak current did not exceed 10% of the CNG current in divalent cation-free solution.
Data Analysis of DoseResponse Relations
Macroscopic currents as a function of cyclic nucleotide concentration were fitted by:
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(1) |
where I is the activated current, Imax is the maximal current, c is the cyclic nucleotide concentration, K1/2 is the cyclic nucleotide concentration activating half of the maximal current, and n is the Hill coefficient.
A similar equation was used to fit the single-channel open probability, Po, as a function of cyclic nucleotide concentration:
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(2) |
where Pmax is the maximal open probability and K1/2 is the cyclic nucleotide concentration giving the half-maximal open probability.
A model for channel activation involving the cooperative binding of cyclic nucleotides to n binding sites, followed by an allosteric closed to open transition, similar to that originally proposed by
where C is the channel in the closed state, cN is the cyclic nucleotide, O is the channel in the open state, Ki are the dissociation constants for the binding sites, and L is the equilibrium "gating" constant, defined as the ratio between open and closed channels. In the limiting case of infinite cooperativity between the binding sites, the above kinetic scheme reduces to (Scheme 2):
with Kd representing the "effective" dissociation constant, defined as Kd = (K1 K2·····Kn-1 Kn)1/n. In this model, the channel open probability, Po, is given by:
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(3) |
where c is the cyclic nucleotide concentration and K1/2 = Kd/(1 + L)1/n. Therefore, K1/2 values are determined by both the apparent ligand affinity for cyclic nucleotides and the probability of the allosteric transition. The maximal open probability, Pmax, is given by L/(1 + L).
The parameters obtained by fitting the single-channel doseresponse data to the Hill equation (Pmax, K1/2, and n) (Equation 2) were used to calculate starting points for the parameters L, Kd, and n for the best fit of Equation 3. Curve fittings were done with IgorPro3.1 (WaveMetrics).
Data Analysis of Current Blockage
Current values in the presence of various concentrations of Ca2+ or Mg2+ were normalized to the current in the absence of divalent cations at a given voltage and plotted versus divalent cation concentration. Data were fitted with the following equation:
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(4) |
where I is the current measured in the presence of the divalent cation concentration d, Imax is the current measured in the absence of divalent cations, Ki is the divalent cation concentration blocking half of Imax, and n is the Hill coefficient.
Equation 4 was fitted to the data for each patch separately and mean values for Ki and n were calculated. All data are given as mean ± SD.
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RESULTS |
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Macroscopic Currents
Currents activated by cGMP or cAMP were studied in excised inside-out patches from oocytes expressing wild-type or individual mutant channels. Fig 1A and Fig B, shows macroscopic currents measured at voltage pulses between -80 and +80 mV and activated by cGMP (A) or by cAMP (B) concentrations producing maximal currents.
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In wild-type channels, maximal currents activated by cGMP or cAMP have very similar values; whereas, quite surprisingly, we found that in the E340G mutant, the current activated by cAMP was reduced to 50% of the current activated by cGMP. Mutation of E340 to aspartate, asparagine, or glutamine had much smaller effects on maximal currents activated by cAMP (see Table 1).
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In Fig 1 C, the modification of the currentvoltage relations induced by mutations of E340 is shown. The mean ratio between the cAMP-gated current flowing at -80 and +80 mV, I(-80)/I(+80), was 1.12 ± 0.03 (n = 7) in wild-type channels, while it was slightly more inwardly rectifying in the mutated channel E340D, with a ratio of 1.30 ± 0.08 (n = 8) and became progressively outwardly rectifying with ratios of 0.69 ± 0.12 (n = 8) in the E340G, 0.51 ± 0.10 (n = 6) in the E340N, and 0.18 ± 0.05 (n = 5) in the E340Q mutants. Rectification ratios were similar for channels activated by cAMP or by cGMP (Table 1). Whether changes of the currentvoltage dependence were due to a modification of the channel permeation or gating properties caused by the mutations will be investigated later (see Fig 4).
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Normalized doseresponse relations at -80 mV are shown in Fig 1D and Fig E. Currents for each channel type were measured from the same patch, plotted versus the concentration of cGMP or cAMP, fitted by the Hill equation (Equation 1), and normalized to the maximal current measured in cGMP at -80 mV. For all mutants, the doseresponse relations were shifted towards higher cyclic nucleotide concentrations with respect to the wild-type channel (Table 1). At -80 mV, K1/2 for cGMP increased from an average value of 2.5 ± 0.5 µM (n = 7) for the wild-type to 15.5 ± 1.6 µM (n = 3) for the E340G mutant, and K1/2 for cAMP increased from an average value of 73 ± 19 µM (n = 7) for the wild-type to 430 ± 66 µM (n = 5) for the E340G mutant. Moreover, the maximal current of the E340G mutant activated by cAMP was 0.44 ± 0.05 (n = 8) of that activated by cGMP. Other mutations affected the current ratio less (Table 1).
Therefore, mutations of glutamate 340 changed the voltage dependence of currents and modified the channel activation by increasing K1/2 and decreasing the ratio between maximal currents activated by cAMP and cGMP.
Single-Channel Properties
To interpret properties of the macroscopic currents in terms of permeation and gating behavior, currents from patches containing only one single olfactory CNG channel were recorded.
Fig 2A and Fig B, shows recordings at -80 mV from a patch containing a single E340G channel. The open probability was plotted in Fig 2 C as a function of cyclic nucleotide concentrations and fitted by the Hill equation (Equation 2). At -80 mV, the average K1/2 was 14 ± 5 µM (n = 4) for cGMP and 400 ± 214 µM (n = 4) for cAMP. These values are very similar to those obtained from macroscopic doseresponse relations (see Table 1).
In the presence of cGMP, the average maximal open probability, Pmax, for E340G channels was 0.79 ± 0.06 (n = 9), whereas, in the presence of cAMP, Pmax was only 0.27 ± 0.12 (n = 9). These values were much lower than Pmax for the wild-type channel, 0.996 ± 0.001 (n = 3) with cGMP and 0.985 ± 0.004 (n = 5) with cAMP (data not shown). The open probability was largely independent of voltage (see Table 2).
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Therefore, the maximal open probability measured in the E340G mutant was significantly lower than that of the wild-type channel and its value depended on which cyclic nucleotide activated the channel.
Single-channel properties were also investigated in other mutants. Single-channel currents from the mutant E340Q could not be measured, probably because of their small amplitude. Assuming the existence of only one open state, an estimate of the current amplitude was obtained by the relation between current variance and mean amplitude of macroscopic currents at various cGMP concentrations (data not shown). The estimated single-channel current was 0.1 pA at +80 mV and 0.03 pA at -80 mV, with a maximal open probability of 0.9.
Fig 3 A shows recordings from a patch containing one single E340N mutant channel obtained at two cGMP concentrations at +80 and -80 mV.
Two conductance levels were present. The low conductance state had a current amplitude of 1 pA at +80 mV. From the low conductance state, the channel made brief transitions to a higher conductance state of
2.5 pA. Occasionally, the current trace returned to baseline from the low conductance state. Transitions to the high conductance state were never observed from the baseline, but only from the low conductance state. At a low cGMP concentration (3 µM), the channel spent most of the time in the closed state, from which it opened to the low conductance state, and very rarely to the high conductance state, as shown in the amplitude histogram of Fig 3 B. At a high cGMP concentration (100 µM), the channel spent most of the time in the low conductance state from which it made more frequent but brief transitions to the high conductance state (Fig 3 C).
The low conductance state was outwardly rectifying [i = -0.5 ± 0.1 pA (n = 5) at -80 mV, and 1.0 ± 0.2 pA (n = 6) at +80 mV], whereas the high conductance state had similar values at both voltages (2.5 pA). Similar results were obtained when the E340N channel was activated by cAMP. However, from single-channel doseresponse curves (data not shown, see Table 2), it was found that the maximal open probability of the low conductance state for cAMP at -80 mV was 0.88 ± 0.02 (n = 2), lower than that for cGMP, which was 0.96 ± 0.01 (n = 3).
Therefore, the E340N mutation modifies the gating properties of the channel by introducing a new conductance state and by reducing the efficacy of activation by cyclic nucleotides more so for cAMP than cGMP.
Voltage Dependence of Permeation Properties
To investigate whether changes in the voltage dependence of the macroscopic currents recorded from various mutants (Fig 1 A) were due to effects of voltage on the permeation or on the gating process, the I-V relations of macroscopic and single-channel currents for wild-type and mutant channels were compared in Fig 4. Macroscopic currents were normalized to their respective values measured at +80 mV, and the values of single-channel and normalized macroscopic currents were averaged from several patches and plotted as a function of voltage. For the mutant E340N, only the single-channel current value of the low conductance state, in which the channel spent most of its time, is shown. Among the different channel types, the amplitude and rectification properties of single-channel current varied; however, they did not depend on which cyclic nucleotide, cAMP or cGMP, activated the channel (data not shown). At -80 mV, the average single-channel current was -3.2 pA in the wild-type, -4.8 pA in the mutated channel E340D, -2.6 pA in the E340G, and -0.5 pA for the low-conductance state in the E340N (see Table 2). The mean ratio between the single-channel current at -80 and +80 mV was 1.07 in the wild type, 1.26 in the mutated channel E340D, 0.72 in the E340G, and 0.5 for the low conductance state in the E340N.
The superimposition of the single-channel I-V curve with the macroscopic I-V curve for the various channel types indicates that the changes in the rectification properties in different mutants are mainly caused by changes in the voltage dependence of the permeation process, rather than by changes in gating (see also Table 2 for values of Pmax at positive and negative voltages).
Gating Model for Channel Activation
The single-channel dose responses of wild-type and mutant channels were plotted in Fig 5, along with the best fit obtained by Equation 3. In the presence of cGMP, Kd had similar values: 29 ± 10 µM (n = 3) wild-type and 31 ± 15 µM (n = 3) for E340D, and 24 ± 12 µM (n = 4) for E340G. On the other hand, L varied from 334 ± 82 for the wild-type to 66 ± 15 for E340D and 5.2 ± 1.3 for E340G mutation. In the presence of cAMP, Kd also had similar values: 333 ± 96 µM (n = 3) for wild-type and 558 ± 120 µM (n = 3) for E340D and 479 ± 241 µM (n = 4) for E340G, though these values differ from those obtained for cGMP. L for cAMP varied from 70 ± 22 for the wild type to 12 ± 7 for E340D and 0.6 ± 0.3 for E340G mutant channels.
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Therefore, mutant channels have Kd values similar to those estimated for the wild-type channel, but are characterized by very different values of the gating constant L, which determines the value for the maximal open probability. Two major conclusions can be drawn from these results. First, mutations at position 340 do not affect the "effective" dissociation constant (Kd) for cAMP and cGMP. Second, mutations at position 340 modify the opening efficiency of the fully liganded channel. Thus, changes in the value of K1/2 in the various mutants originate from changes of L.
Divalent Cation Blockage
It is well known from previous work on the subunit of the bovine retinal rod CNG channel that E363 controls channel blockage by extracellular divalent cations. We investigated here the role on such blockage of the residue E340 at the corresponding position in the olfactory CNG channel.
First, we measured the macroscopic doseresponse relations for currents activated by cAMP or cGMP in inside-out patches from wild-type, E340G, and E340N channels in the presence of 1 mM Ca2+ in the patch pipette. The presence of 1 mM external Ca2+ did not significantly affect K1/2 for either cyclic nucleotide (data not shown), indicating that external Ca2+ does not modify the gating properties of these channels (see also Fig 7).
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The blocking effect of external Ca2+ and Mg2+ was therefore investigated by measuring the I-V relations of wild-type and mutant CNG channels in the presence of increasing Ca2+ or Mg2+ concentrations at the extracellular side of outside-out patches.
Fig 6A and Fig B, (left) shows the I-V relations for wild-type channels in which increasing concentrations of external Ca2+ (A) or Mg2+ (B) progressively reduced both the inward and outward currents in a voltage-dependent manner. When glutamate 340 was replaced by a neutral asparagine or glycine residue, blockage by external divalent cations was greatly reduced, and even high concentrations of external Ca2+ or Mg2+, 510 mM, only partially blocked the current (Fig 6).
To investigate whether an acidic residue at position 340 was crucial for Ca2+ and Mg2+ blockage, the charge-conserving substitution E340D was analyzed. Fig 6 shows that external divalent cations powerfully blocked E340D channels, and that the voltage dependence of the blockage by external Ca2+ differed from that observed in wild-type channels. Indeed (see also Table 3), the blocking effect at positive voltages was more pronounced in the E340D mutant than in wild-type channels (see DISCUSSION).
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The concentration dependence of extracellular Ca2+ and Mg2+ blockage is better illustrated in Fig 6CF. Current values from the experiments shown in Fig 6A and Fig B, measured at +80 or -80 mV in the presence of various concentrations of Ca2+ or Mg2+ were normalized to the current in the absence of divalent cations and plotted versus divalent cation concentration. For external divalent cation blockage at various voltages, Ki was calculated from the best fit of Equation 4 to the data (see Table 3). At -80 mV for Ca2+, Ki was found to increase from a similar average value of 47 ± 21 µM (n = 6) for wild-type and 47 ± 7 µM (n = 6) for E340D, to 6.3 ± 2.5 mM (n = 6) for E340G and 6.5 ± 4.0 mM (n = 4) for E340N mutant channels. At -80 mV for Mg2+, Ki increased from an average value of 70 ± 34 µM (n = 11) for wild-type and 53 ± 17 µM (n = 4) for E340D, to 1.1 ± 0.3 mM (n = 7) for E340G and 17.0 ± 7.2 mM (n = 4) for E340N mutant channels.
The blockage by external divalent cations was also found to be relieved in the E340Q mutant channels (data not shown), with average values reported in Table 3. Hill coefficients for wild type and E340D were always close to one, indicating that one divalent cation is likely sufficient to block the pore in these channels. A 1,000-fold higher external divalent concentration was necessary to block 50% of the current in mutant channels with a neutral residue in position 340, and Hill coefficients were below unity.
The blockage by internal Ca2+ of wild-type and mutant channels was also investigated. Macroscopic currents in inside-out patches were activated by 1 mM cGMP in the presence of various Ca2+ concentrations at the cytoplasmic side of the patch (data not shown), and the average Ki for internal Ca2+ blockage at +80 mV was found to be 1.0 ± 0.7 mM (n = 5) for wild-type and 1.5 ± 0.4 mM (n = 4) for E340G mutant channels. Similar results were also found for the E340N mutation. Therefore, charge neutralization of E340 does not significantly modify the blockage by intracellular Ca2+ of the olfactory CNG channel.
These results show that the presence of an acidic residue at position 340 of the bovine olfactory channel is an important determinant of blockage by external divalent cations.
Single-Channel Analysis of External Ca2+ Blockage
To investigate whether external divalent cation blockage on macroscopic current is due to a change in permeation and/or gating properties of the olfactory CNG channel, single-channel properties were examined. Fig 7 A shows recordings from a patch in the outside-out configuration containing wild-type channels activated by 5 µM cGMP at -80 mV. At saturating cGMP concentrations, wild-type channels are open most of the time, making it difficult to estimate the single-channel amplitude reliably, so a lower concentration of cGMP was used for estimating the single-channel amplitude. The addition of 50 µM external Ca2+ caused a partial blockage of the single-channel current and in the presence of 1 mM Ca2+, no current was detected at -80 mV.
When the same experiments were repeated with a patch containing a single E340G mutant channel (Fig 7 B), activated by 1 mM cGMP, the single-channel amplitude was only slightly reduced in the presence of 1 or even 10 mM external Ca2+.
Single-channel currents from the patches shown in Fig 7A and Fig B, in the presence of increasing concentrations of extracellular Ca2+, were estimated from amplitude histograms. The obtained values were normalized to the single-channel current measured in the absence of divalent cations and plotted as a function of Ca2+ concentration at +80 and -80 mV (see Fig 7C and Fig D). The best fit of Equation 4 to the data (continuous lines) gave a value of Ki for Ca2+ at +80 mV of 250 µM for wild-type and 32 mM for E340G, while at -80 mV, Ki for Ca2+ was 31 µM for wild-type and 8.8 mM for E340G. Similar results were obtained in two other patches for each channel type.
A comparison between these results on single-channel current and those on macroscopic currents (Table 3) shows that Ki and n values are in good agreement, indicating that external Ca2+ blockage diminishes the single-channel amplitude, affecting permeation rather than gating.
Divalent Cation Permeation
The permeation of divalent cations was investigated in outside-out patches by substituting external NaCl (110 mM) with equiosmolar concentrations (73.3 mM) of CaCl2 or MgCl2. Channels were activated by 1 mM cGMP in the patch pipette and voltage steps of 150-ms duration from a holding potential of 0 mV were given from -80 to +80 mV in 20-mV steps. Fig 8A and Fig B, shows recordings from wild-type and E340G mutants, respectively. I-V relations were plotted in Fig 8C and Fig D.
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From these experiments, the ratio between the current carried by external Ca2+ and Na+ ions, ICa/INa, at -80 mV was calculated. For wild-type channels, the average value of ICa/INa at -80 mV was 0.049 ± 0.004 (n = 4) and increased to 0.16 ± 0.03 (n = 6) for E340G mutant channels. The average reversal potential, Vrev, under bi-ionic conditions (internal Na and external divalent cations) was 25 ± 6 mV (n = 8) for Ca2+ and 45 ± 7 mV (n = 5) for Mg2+ for wild-type channels. For E340G mutant channels, Vrev was 24 ± 5 mV (n = 5) for Ca2+ and 45 ± 15 mV (n = 4) for Mg2+.
These results show that reversal potentials and therefore permeability ratios, were not affected by the E340G mutation. However, the E340G mutation caused an increase of the inward Ca2+ current relatively to the Na+ current.
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DISCUSSION |
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The results presented in this paper demonstrate that the residue at position 340 of the bovine olfactory CNG channel affects permeation and gating properties in the absence of divalent cations and controls the efficacy of blockage by external Ca2+ and Mg2+ ions. Moreover, our findings highlight similarities and differences with the role played by the corresponding residue 363 of the bovine rod CNG channel.
Gating by cAMP and cGMP
It is well established that cGMP and cAMP elicit similar maximal responses in wild-type olfactory channels, whereas K1/2 for cGMP is several-fold lower than that for cAMP. All mutant channels presented in this study had higher K1/2 values compared with wild-type channels, and showed a decrease of the maximal current activated by cAMP with respect to cGMP, especially in the E340G mutant. The explanation of these results is not obvious: the decrease in cAMP maximal current could derive either from a reduced single-channel amplitude or from different gating properties of CNG channels when activated by cAMP or cGMP. Moreover, the shift in K1/2 could originate from a modification in the binding reactions for cyclic nucleotides (i.e., Kd) or the subsequent allosteric rearrangement leading to channel opening (i.e., L) or from both effects.
Single-channel analysis allowed us to distinguish between these possible explanations. In all tested mutants, the single-channel conductance activated by cAMP and cGMP was the same, but open probabilities were different: in the E340G mutant Pmax did not exceed 50% of that observed with cGMP. Moreover, analysis of the data by using an allosteric model similar to that used by
Permeation
We found that mutation of E340 caused a modification in the I-V relations. The analysis of single-channel activity (Table 2) showed that the rectification of the I-V curves is primarily determined by the voltage dependence of ion permeation rather than of the gating process. This result differs from what was previously measured in retinal rod. Indeed,
Two conductance levels were observed in the E340N mutant. Although it was outside the scope of our study to develop a kinetic model explaining these multiple conductance levels, this result further shows that the bovine olfactory CNG channel has single-channel properties different from those of the catfish olfactory CNG channel. Indeed, the CNG channel from catfish has multiple conductance states (
These results show that channels from different species could have some specific properties that are not necessarily shared by the majority of CNG channels, despite belonging to the same class.
Divalent Cation Blockage
Ca2+ and Mg2+ block the current carried by Na+ ions through olfactory CNG channels from the extracellular side and to a lesser extent from the intracellular side. The external divalent cation blockage was drastically reduced in mutant channels where E340 was replaced by a neutral amino acid (Table 1), while internal divalent blockage was largely unaffected. These findings are in agreement with similar results obtained with mutations at position E363 of the subunit from bovine retinal rods (
A special case is the E340D mutant. The charge-conserving mutation E340D preserved the external divalent cation blockage, but altered its voltage dependence with a more pronounced effect for Ca2+ than for Mg2+ (Table 3). Indeed, we unexpectedly found that external Ca2+ blockage was more effective at positive (Ki = 18 µM at +80 mV) than negative (Ki = 47 µM at -80 mV) voltages. In the E363D mutant from retinal rods (
How can the different voltage dependence between Ca2+ and Mg2+ blockage in the olfactory E340D channel be explained? A similar result has also been previously observed in the blockage by internal divalent cations in native CNG channels from retinal rods (
From a molecular point of view, these differences could have several origins. Although glutamate and aspartate have the same charge, they have a different length of the amino acid side chains and the protonation of aspartate could be different from that of glutamate. Alternatively, the binding site may change its location within the transmembrane electrical field, and/or the stereochemical coordination of divalent cations may be altered.
Comparison with Native Channels
The voltage dependence of blockage in 3 homomers and native channels is largely similar in that blockage was more pronounced at negative than at positive potentials. However, the values for Ki were different. The Ki for external Ca2+ blockage in native olfactory CNG channels from the frog Rana esculenta was found to be 265 µM at -70 mV and
2 mM at +80 mV (see Figure 6 B of
3 homomers we found 47 µM at -80 mV and 383 µM at +80 mV. These different Ki values could originate from the different subunit composition of native channels: indeed, native olfactory CNG channels from the olfactory sensory neurons of the rat are composed by the coassembly of the three different types of subunits
3,
4, and ß1b (
3 subunit of bovine olfactory CNG channels. These residues are likely to play the key role in the blockage by external divalent cations, since we have shown that mutation of glutamate at position 340 of the
3 subunit into aspartate or glycine changes the sensitivity to external Ca2+ and Mg2+ by more than two orders of magnitude.
Recent experiments by 3 subunit.
and ß subunits, and not on channels formed from
subunits alone. Whether the additional olfactory subunits are responsible for the reduced sensitivity to cAMP induced by external Ca2+ in native olfactory channels remains to be determined.
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Footnotes |
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1 Abbreviation used in this paper: CNG channel, cyclic nucleotidegated channel.
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Acknowledgements |
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We thank V. Torre, F. Gambale and A. Miri for comments on the manuscript and F. Pittaluga, G. Boido, G. Gaggero, P.G. Gagna, D. Magliozzi, and P. Guastavino for technical assistance. L. Giovanelli checked the English. Special thanks to B. Norcio for many helpful discussions.
This work was supported by European Community contract BIO4-CT96-0593 (U.B. Kaupp and A. Menini) and a grant from the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen IVA-10201095 (U.B. Kaupp).
Submitted: 15 May 2000
Revised: 26 June 2000
Accepted: 27 June 2000
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References |
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Becchetti, A., Gamel, K., and Torre, V. 1999. Cyclic nucleotidegated channels. Pore topology studied through the accessibility of reporter cysteines. J. Gen. Physiol. 114:377-392
Bönigk, W., Bradley, J., Müller, F., Sesti, F., Boekhoff, I., Ronnett, G.V., Kaupp, U.B., and Frings, S. 1999. The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits. J. Neurosci. 13:5332-5347.
Bradley, J., Li, J., Davidson, N., Lester, H.A., and Zinn, K. 1994. Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP. Proc. Natl. Acad. Sci. USA. 91:8890-8894[Abstract].
Brown, R.L., Geber, W.V., and Karpen, J.W. 1993. Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photoaffinity analog 8-p-azidophenacylthio-cGMP. Proc. Natl. Acad. Sci. USA. 90:5369-5373[Abstract].
Brown, R.L., Snow, S.D., and Haley, T.L. 1998. Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels. Biophys. J. 75:825-833
Bucossi, G., Nizzari, M., and Torre, V. 1997. Single-channel properties of ionic channels gated by cyclic nucleotides. Biophys. J. 72:1165-1181[Abstract].
Colamartino, G., Menini, A., and Torre, V. 1991. Blockage and permeation of divalent cations through the cyclic GMP-activated channel from tiger salamander retinal rods. J. Physiol. 440:189-206[Abstract].
Dhallan, R.S., Yau, K.-W., Schrader, K.A., and Reed, R.R. 1990. Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature. 347:184-187[Medline].
Dzeja, C., Hagen, V., Kaupp, U.B., and Frings, S. 1999. Ca2+ permeation in cyclic nucleotide-gated channels. EMBO (Eur. Mol. Biol. Organ.) J. 18:131-144
Eismann, E., Muller, F., Heinemann, S.H., and Kaupp, U.B. 1994. A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity. Proc. Natl. Acad. Sci. USA. 91:1109-1113[Abstract].
Fabiato, A. 1988. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 157:378-417[Medline].
Finn, J.T., Grunwald, M.E., and Yau, K.W. 1996. Cyclic nucleotide-gated ion channels: an extended family with diverse functions. Annu. Rev. Physiol. 58:395-426[Medline].
Fodor, A.A., Black, K.D., and Zagotta, W.N. 1997. Tetracaine reports a conformational change in the pore of cyclic nucleotidegated channels. J. Gen. Physiol. 110:591-600
Frings, S., Seifert, R., Godde, M., and Kaupp, U.B. 1995. Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels. Neuron. 15:169-179[Medline].
Gavazzo, P., Picco, C., and Menini, A. 1997. Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells. Proc. R. Soc. Lond. B Biol. Sci. 264:1157-1165[Medline].
Gordon, S.E., and Zagotta, W.N. 1995. Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels. Neuron 14:857-864[Medline].
Gordon, S.E., Varnum, M.D., and Zagotta, W.N. 1997. Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotide-gated channels. Neuron. 19:431-441[Medline].
Goulding, E.H., Ngai, J., Kramer, R.H., Colicos, S., Axel, R., Siegelbaum, S.A., and Chess, A. 1992. Molecular cloning and single channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons. Neuron. 8:45-58[Medline].
Goulding, E.H., Tibbs, G.R., and Siegelbaum, S.A. 1994. Molecular mechanism of cyclic-nucleotide-gated channel activation. Nature. 372:369-374[Medline].
Hackos, D.H., and Korenbrot, J.I. 1999. Divalent cation selectivity is a function of gating in native and recombinant cyclic nucleotidegated ion channels from retinal photoreceptors. J. Gen. Physiol. 113:799-817
Herlitze, S., and Koenen, M. 1990. A general and rapid mutagenesis method using polymerase chain reaction. Gene. 91:143-147[Medline].
Kaupp, U.B., Niidome, T., Tanabe, T., Terada, S., Bönigk, W., Stühmer, W., Cook, N.J., Kangawa, K., Matsuo, H., and Hirose, T. et al. 1989. Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature. 342:762-766[Medline].
Kaupp, U.B. 1995. Family of cyclic nucleotide gated ion channels. Curr. Opin. Neurobiol. 5:434-442[Medline].
Kleene, S.J. 1995. Block by external calcium and magnesium of the cyclic nucleotide-activated current in olfactory cilia. Neuroscience. 66:1001-1008[Medline].
Kleene, S.J. 1999. Both external and internal calcium reduce the sensitivity of the olfactory cyclic nucleotide-gated channel to cAMP. J. Neurophysiol 81:2675-2682
Liman, E.R., and Buck, L.B. 1994. A second subunit of the olfactory cyclic nucleotide-gated channel confers high sensitivity to cAMP. Neuron. 13:611-621[Medline].
Liu, M., Chen, T.Y., Ahamed, B., Li, J., and Yau, K.W. 1994. Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. Science. 266:1348-1354[Medline].
Melton, D.A., P.A. Krieg, M.R. Rebagliati, Maniatis, T.K. Zinn, and M.R. Green. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acid Res. 12:70357056.
Menini, A., and Nunn, B.J. 1990. The effect of pH on the cyclic GMP-activated conductance in retinal rods. In Borsellino A., Cervetto L., Torre V., eds. Sensory Transduction. New York, NY, Plenum Publishing Corp, 175-181.
Menini, A. 1995. Cyclic nucleotide-gated channels in visual and olfactory transduction. Biophys. Chem. 55:185-196[Medline].
Menini, A. 1999. Calcium signalling and regulation in olfactory neurons. Curr. Opin. Neurobiol. 9:419-426[Medline].
Nakamura, T., and Gold, G.H. 1987. A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature. 325:442-444[Medline].
Paoletti, P., Young, E.C., and Siegelbaum, S.A. 1999. C-linker of cyclic nucleotide-gated channels controls coupling of ligand binding to channel gating. J. Gen. Physiol. 113:17-33
Park, C.S., and MacKinnon, R. 1995. Divalent cation selectivity in a cyclic nucleotide-gated ion channel. Biochemistry. 34:13328-13333[Medline].
Perry, R.J., and McNaughton, P.A. 1991. Response properties of cones from the retina of the tiger salamander. J. Physiol. 433:561-587[Abstract].
Picones, A., and Korenbrot, J.I. 1995. Permeability and interaction of Ca with cGMP-gated ion channels differ in retinal rod and cone photoreceptors. Biophys. J 69:120-127[Abstract].
Root, M.J., and MacKinnon, R. 1993. Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. Neuron. 11:459-466[Medline].
Root, M.J., and MacKinnon, R. 1994. Two identical noninteracting sites in an ion channel revealed by proton transfer. Science. 265:1852-1856[Medline].
Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74:5463-5467[Abstract].
Sautter, A., Zong, X., Hofmann, F., and Biel, M. 1998. An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta subunit expressed in olfactory neurons. Proc. Natl. Acad. Sci. USA. 95:4696-4701
Scott, S.P., and Tanaka, J.C. 1998. Three residues predicted by molecular modeling to interact with the purine moiety alter ligand binding and channel gating in cyclic nucleotide-gated channels. Biochemistry. 37:17239-17252[Medline].
Seifert, R., Eismann, E., Ludwig, J., Baumann, A., and Kaupp, U.B. 1999. Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates. EMBO (Eur. Mol. Biol. Organ.) J. 18:119-130
Sun, Z.-P., Myles, H.A., Goulding, E.H., Karlin, A., and Siegelbaum, S.A. 1996. Exposure of residues in cyclic nucleotide-gated channel pore: P region structure and function in gating. Neuron 16:141-149[Medline].
Sunderman, E.R., and Zagotta, W.N. 1999a. Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels. J. Gen. Physiol. 113:601-619
Sunderman, E.R., and Zagotta, W.N. 1999b. Sequence of events underlying the allosteric transition of rod cyclic nucleotidegated channels. J. Gen. Physiol. 113:621-640
Tibbs, G.R., Goulding, E.H., and Siegelbaum, S.A. 1997. Allosteric activation and tuning of ligand efficacy in cyclic nucleotide-gated channels. Nature. 386:612-615[Medline].
Varnum, M.D., Black, K.D., and Zagotta, W.N. 1995. Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels. Neuron. 15:619-625[Medline].
Varnum, M.D., and Zagotta, W.N. 1997. Interdomain interactions underlying activation of cyclic nucleotide gated channels. Science 278:110-113
Zagotta, W.N., and Siegelbaum, S.A. 1996. Structure and function of cyclic nucleotide-gated channels. Annu. Rev. Neurosci. 19:235-263[Medline].
Zimmerman, A.L. 1995. Cyclic nucleotide-gated channels. Curr. Opin. Neurobiol. 5:296-303[Medline].
Zimmerman, A., and Baylor, D.A. 1992. Cation interactions within the cyclic GMP-activated channel of retinal rods from the tiger salamander. J. Physiol. 449:759-783[Abstract].
Zimmerman, A., Karpen, J.W., and Baylor, D.A. 1988. Hindered diffusion in excised membrane patches from retinal rods outer segments. Biophys. J. 54:351-355[Abstract].
Zong, X., Zucker, H., Hofmann, F., and Biel, M. 1998. Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channel. EMBO (Eur. Mol. Biol. Organ.) J. 17:353-362