Correspondence to: Zhe Lu, University of Pennsylvania, Department of Physiology, D302A Richard Building, 3700 Hamilton Walk, Philadelphia, PA 19104. Fax:215-573-1940 E-mail:zhelu{at}mail.med.upenn.edu.
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Abstract |
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Polyamines block the retinal cyclic nucleotide-gated channel from both the intracellular and extracellular sides. The voltage-dependent mechanism by which intracellular polyamines inhibit the channel current is complex: as membrane voltage is increased in the presence of polyamines, current inhibition is not monotonic, but exhibits a pronounced damped undulation. To understand the blocking mechanism of intracellular polyamines, we systematically studied the endogenous polyamines as well as a series of derivatives. The complex channel-blocking behavior of polyamines can be accounted for by a minimal model whereby a given polyamine species (e.g., spermine) causes multiple blocked channel states. Each blocked state represents a channel occupied by a polyamine molecule with characteristic affinity and probability of traversing the pore, and exhibits a characteristic dependence on membrane voltage and cGMP concentration.
Key Words: retinal cyclic guanine monophosphategated channel, ion permeation, protonation, polyamine, diamine
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INTRODUCTION |
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Cyclic nucleotidegated (CNG)1 channels are a class of nonselective cation channels present in many tissue types. CNG channels open when cAMP and/or cGMP binds to a cyclic nucleotidebinding motif formed by part of the COOH-terminal segment of the channel polypeptide chain (
For more than a decade, the CNG channel has been known to be blocked by divalent cations such as Ca2+ and Mg2+ (
Extracellular polyamine block of CNG channels is very similar to intracellular polyamine block of glutamate-gated and ACh-receptor channels (e.g., compare
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METHODS |
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Molecular Biology and Oocyte Preparation
The cDNA of the bovine rod cGMP-gated channel subunit cloned into pGEM-HE plasmid was kindly provided by Dr. Steven A. Siegelbaum (
Patch Recording and Data Analysis
The CNG channel currents were recorded in the inside-out configuration from the membrane of Xenopus oocytes (injected with the CNG channel cRNA) with an Axopatch 200B amplifier (Axon Instruments, Inc.). The recorded signal was filtered at 1 kHz and sampled at 5 kHz using an analogue-to-digital converter (DigiData 1200; Axon Instruments, Inc.) interfaced with a personal computer. pClamp6 software (Axon Instruments, Inc.) was used to control the amplifier and acquire the data. Macroscopic currentvoltage curves were recorded as membrane voltage was linearly ramped (25 mV/s). The currents obtained in the absence of cGMP were used as templates for subsequent off-line background current corrections. All curve fittings were carried out using Origin software version 5 (Microcal Software, Inc.).
Recording Solutions
Both the intracellular and extracellular solutions contained (mM) 130 NaCl, 0.5 EDTA, and 10 HEPES, pH 7.6 or 8.6, as specified. Unless specified otherwise, to activate the channel, 1 mM cGMP was included in the intracellular solution. The intracellular solutions containing diamines, polyamines, and philanthotoxin-343 (PhTx) were prepared daily.
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RESULTS |
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Effect of a Pore Mutation on CNG Channel Block by Spermine
Fig 1 A shows the macroscopic currentvoltage relationship of the wild-type retinal CNG channel in the absence and presence of various concentrations of intracellular spermine. The data were recorded in the inside-out configuration by ramping the membrane voltage from -80 to +80 mV. In the absence of blocking ions, the I-V curve is nearly linear. Spermine in the intracellular solution inhibits the current in a voltage-dependent manner.
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Fig 1 B shows the I-V curves of the E363G mutant channel in the absence and presence of various concentrations of spermine. As previously reported, glycine substitution for residue E363 dramatically reduces the inward current (
Fig 1C and Fig D, shows the fractions of unblocked wild-type and mutant CNG currents as a function of voltage, in the presence of various concentrations of spermine. Wild-type channel block by intracellular polyamine varies with membrane voltage in a complex manner: it increases when the membrane voltage is increased from -80 to -20 mV, and then decreases as the voltage approaches +25 mV, and increases again when the voltage is further increased. Consequently, the blocking curves shown in Fig 1 C display a minimum followed by a maximum. Fig 1 D shows that E363G mutation merely shifts the spermine-blocking curves by approximately +40 mV without altering their general multiphasic appearance, as if residue E363 (located at the external end of the ion conduction pore) affects spermine block by a through-space electrostatic effect. This observation supports the idea that intracellular spermine inhibits the CNG channel by acting as a pore blocker.
Comparison of CNG Channel Block by Intracellular Spermine and Putrescine
Fig 2A and Fig C, compares the effects of spermine and putrescine on the I-V curve of the wild-type CNG channel. Fig 2B and Fig D, plots the corresponding fractions of unblocked current as a function of membrane voltage between -80 and +80 mV. As discussed above, the spermine-blocking curve in Fig 2 B has a minimum followed by a maximum. In contrast, the putrescine-blocking curve has only a minimum, although it appears to reach a plateau at +80 mV (Fig 2 D;
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Block of the CNG Channel by a Series of Diamines
To learn how methylene groups affect the blocking behavior of polyamines, we examined CNG channel block by a series of putrescine analoguesdiamines with methylene (CH2) chains of varying length between the two amine groups. To obtain a more complete picture of their blocking behaviors, we further increased the voltage range to between -180 and +180 mVthe widest range within which we could collect sufficient data before the oocyte membrane ruptured. Fig 3 shows the I-V curves of the CNG channels in the absence and presence of several concentrations of nine diamines, denoted DMC2 through DMC10, that contain from 2 to 10 methylene groups. With the exception of DMC2, the inhibitory potency of diamines increases with each additional methylene group. All the diamines blocked the channel in a voltage-dependent manner. The voltage dependence of channel block by various diamines is more clearly shown in Fig 4, in which we plotted the fraction of unblocked current in the presence of each diamine as a function of membrane voltage. The blocking curves corresponding to various diamines are quite different. Those for DMC2, DMC3, DMC6, and DMC7 have mainly two phases: a descending phase followed by an ascending phase. As discussed earlier, the curve for DMC4 (putrescine) has a very prominent second descending phase, and consequently shows a minimum followed by a maximum. Interestingly, for DMC5, the minimum and maximum seem to merge into an extended plateau that precedes a second descending phase. The curves corresponding to DMC8 through DMC10 simply approach a nonzero level at the end of the experimentally accessible voltage range.
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Block of the CNG Channel by Spermine at Two Different Intracellular pH
To gain insight into the effect of polyamine charge on channel block, we altered the average number of charged amines in spermine by adjusting intracellular pH, exploiting the fact that the pKa for some amine groups is in the range of 8 to 9 (
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Block of the CNG Channel by Spermine and Spermidine Over a Wider Voltage Range
Fig 6A and Fig B, shows the I-V curves of the CNG channel in the absence or presence of spermine and spermidine, while C and D shows the fractions of current not blocked by spermine and spermidine, respectively, against membrane voltage. Over this much wider range of membrane voltage (-180 to +180 mV), the curve for spermine now displays two pairs of minima and maxima. Although the multiple phases in the curves for spermidine are not as well defined (Fig 6 D), the curve for 100 µM spermidine has a small "minimum" (indicated by an arrow) at voltages where the second minimum in the spermine curve occurs (compare C with D). We have repeated this measurement many times and the small "minimum" in the 100 µM spermidine curve was invariably observed. Thus, the blocking curves for spermidine and spermine are likely fundamentally similartwo pairs of minima and maxima at comparable membrane voltagesalthough the multiple phases are poorly separated in the case of spermidine.
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Block of the CNG Channel by Spermine at Various Concentrations of cGMP
To test whether block of the CNG channel by polyamines depends on cGMP concentration, we examined channel block by spermine at various concentrations of cGMP. Fig 7AF, shows the I-V curves for the channel in the absence and presence of various concentrations of spermine, as the concentration of cGMP was varied from 10 µM to 1 mM (K1/2 for cGMP is ~80 µM). In the presence of spermine, the shape of the I-V curves at positive membrane voltages varied significantly with the concentration of cGMP. The variations are more clearly shown in Fig 7GL, which shows the fractions of unblocked current as a function of membrane voltage. The first descending and ascending phases are very similar at all cGMP concentrations tested. The second descending phase became steeper, and a second ascending phase became apparent, when the cGMP concentration was increased to 30 µM. The third descending phase did not appear until cGMP was increased to 50 µM. In other words, the first minimum and maximum pair was present at all concentrations of cGMP, but the second minimum and maximum pair at positive voltages, absent at 10 µM cGMP, gradually appeared when cGMP concentration was raised.
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Block of the CNG Channel by a Spermine Derivative
To test whether depolarization-induced relief of channel block results from spermine permeation, we studied the blocking behavior of a natural derivative of spermine isolated from a spider venom, called PhTx. PhTx can be thought of as spermine with a bulky group attached to one end. Fig 8 A shows the I-V curves of the channel in the absence and presence of PhTx at the concentrations indicated. Unlike the complex I-V curves obtained in the presence of spermine (see Fig 6 A), the I-V curves in the presence of PhTx merely display a downward curvature, as expected for a nonpermeant ionic pore blocker. Fig 8 B shows that the fraction of unblocked current in the presence of PhTx decreases with increasing membrane voltage. No significant voltage-induced relief of channel block by PhTx was observed between -180 to +180 mV.
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DISCUSSION |
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The extent of CNG channel block by intracellular polyamines varies with membrane voltage in a complex manner. For example, when membrane voltage is increased from -80 to +80 mV in the presence of spermine, the CNG current varies in three phases: two descending phases with an intervening ascending phase, resulting in a minimum followed by a maximum in the voltage-dependent blocking curve (Fig 2 B). A similar phenomenon, although occurring over a much wider range of membrane voltage, was also observed with polyamines containing fewer amine groups; e.g., putrescine (Fig 2 F). As proposed previously, the first and second descending phases in the relative current versus membrane voltage plot can be attributed to channel block by a polyamine molecule in one of two conformations with different affinity, while the intervening ascending phase can be accounted for by permeation of the polyamine in its higher affinity conformation and consequent resumption of ionic current (
To test whether the depolarization-induced relief of channel block results from polyamine permeation, we examined channel block by PhTx, a derivative of spermine whose one end is attached to a bulky chemical group. Such a chemical modification was previously shown to dramatically hinder spermine permeation through glutamate-gated channels (
Analysis of Channel Block by Various Diamines
To gain insight into the complex blocking behaviors of diamines with methylene chains of varying length, we analyzed the blocking curves of the various diamines using a model shown in Fig 9 A, similar to one previously used to analyze spermine block of this channel (
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(1) |
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Using rate constants, we obtain:
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(2) |
where [DM] is the intracellular diamine concentration (the extracellular diamine concentration is zero). Assuming the second conformation is nonpermeant (i.e., k-2b = 0), Equation 2 becomes:
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(3) |
where K1b = k-1b /k1b, the equilibrium dissociation constant for the nonpermeant form. Further assuming that all rate constants vary exponentially with membrane voltage, Equation 3 becomes:
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(4) |
or
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(5) |
where K1a = k-1a/k1a is the equilibrium dissociation constant for the permeant form, and quantities zix and Zix are the apparent valences associated with the corresponding constants. Vm is membrane voltage, and F, R, and T have their usual meanings.
Fig 9 B illustrates how the various transitions in the model (or the various constants in Equation 4 or Equation 5) are related to the different phases in a putrescine (DMC4)-blocking curve. Curve a superimposed on the data is a fit of Equation 5, while the other three curves correspond to three hypothetical cases: putrescine acting as (b) a single-conformation, high-affinity, permeant blocker (K1b = ), (c) a single-conformation, high-affinity, nonpermeant blocker (K1b =
and k-2a = 0), or (d) a single-conformation, low-affinity, nonpermeant blocker (K1a =
and k-2b = 0). The values of the corresponding parameters used to generate curves bd are the same as those for the full curve a. Thus, the first and second descending phases of the blocking curve are accounted for by channel block by putrescine in the high- and low-affinity conformations, respectively, while the intervening ascending phase reflects permeation of putrescine in the high-affinity conformation. Since k-2a/k-1a is the relative probability of a diamine bound in the pore escaping to the external solution versus returning to the intracellular solution, it provides a measure of diamine permeation. Quantities k-2a/k-1a and "z-1a + z-2a" were treated as single adjustable parameters in the fit of Equation 5.
To analyze those blocking curves that do not display a significant second descending phase, we omitted the term K1b:
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(6) |
Examples of analyses of diamine data using Equation 5 and Equation 6 are shown in Fig 4; the curves superimposed on the data are all in fact fits of these equations. The parameters obtained from these fits are summarized in Fig 10.
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Fig 10A and Fig B, plot K1a and Z1a versus the number of methylene groups in the tested diamines, respectively. These two parameters are mainly related to the first descending phase; i.e., binding and unbinding of diamines in the permeating conformation. With the exception of DMC2, longer diamines generally have a lower K1a (higher affinity), consistent with the observation that longer diamines have higher blocking potency (Fig 3). Although the amine groups of shorter diamines may both be located inside the narrow region, with increasing methylene chain length the trailing amine group should gradually extend out of the pore into the intracellular solution, as originally proposed for sarcoplasmic reticulum K+ channel block by bis-Q compounds (di-quaternary ammoniums) (
When the number of methylene groups in diamines increases, k-2a/k-1a becomes smaller (Fig 10 C). Longer diamines being less likely to go across the pore to the extracellular side is consistent with the expectation that energy barriers are higher for the longer and more hydrophobic diamines. Since "z-1a + z-2a" decreases with diamine length, the enhancement of permeation by membrane depolarization decreases with diamine length (Fig 10 D). These two factors together account for the observed reduction in permeation for longer diamines.
The blocking curves of both DMC4 and DMC5 have a rather prominent second blocking phase, a behavior apparently associated with methylene chain length between the amine groups. Thus, we used the full Equation 5 to analyze their behavior. We found that the K1b value of DMC5 is significantly lower (high affinity) than that of DMC4 (Fig 10 E), whereas the apparent affinity (or K1a) of the high-affinity conformations of the two diamines is comparable (Fig 10 A). These findings are compatible with the observation that the first and second blocking phases of DMC5 are less well separated. The reduced separation of the two blocking phases and the lower permeability (k-2a/k-1a) of DMC5 together explain why the minimum and maximum that clearly separate the two blocking phases in the case of DMC4 become merged into an extended plateau in the case of DMC5 (Fig 4).
Although various diamines exhibit rather different blocking behaviors, we found that increasing methylene chain length generally favors diamine binding, but reduces the likelihood of permeation. Thus, not surprisingly, hydrophobic forces play a critical role in the interactions between channel and diamines.
Protonation Underlies the Different Blocking Conformations of Polyamines
Ammonia has a pKa value of 9.3. Attaching an alkyl group of arbitrary length to the nitrogen atom raises its pKa to a nearly uniform value around 10.6 (
We surmise that the hypothesized multiple blocking conformations of spermine represent its different protonated states, the more protonated species corresponding to the higher-affinity, more permeant conformation, and the less protonated species corresponding to the lower-affinity, less permeant conformation. If this is true, altering intracellular pH should alter the spermine-blocking curve in the following manner. At a given spermine concentration, raising intracellular pH deprotonates spermine and thus decreases its fraction in the high-affinity form, which should shift the first blocking (descending) phase to more positive membrane voltages. Simultaneously, the increased fraction in the low-affinity (less protonated) form should shift the second blocking (descending) phase to lower voltages. As the two blocking phases are now closer to one another, the amplitude of the intervening ascending phase (reflecting permeation of the high-affinity form) should decrease. The steepness (apparent voltage dependence) of the two blocking phases should be unaffected by pH, because each reflects how the channels interact with a given form of spermine, not its concentration. This is, indeed, what we observed when we raised intracellular pH from 7.6 to 8.6. Fig 5 B plots the fraction of unblocked current in the presence of 10 µM spermine at intracellular pH 7.6 and 8.6. We analyzed the data according to the scheme shown in Fig 11, which assumes that the two spermine conformations reflect the titration of a single amine group. The fraction of unblocked current in the presence of spermine is then given by:
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(7) |
where = 10-pH/(10-pH + 10-pKa). The two smooth curves superimposed on the blocking curves obtained at intracellular pH 7.6 and 8.6 are simultaneous fits of Equation 7; i.e., all the fitting parameters are the same for both curves. The pKa of the titrated amine group, determined from the fit, is 9.1 ± 0.1 (mean ± SEM; n = 3), very close to 8.9, the second lowest pKa value of spermine (
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Although the observed multiple blocked channel states are well accounted for by nonuniform protonation of the blocking agent, they can in principle be equally well accounted for by different protonation states of the channel. In the olfactory CNG channel, protonation of a glutamate residue (equivalent to E363 in the retinal channel), located at the external end of the pore, reduces the single-channel conductance (
Additional Blocking Components of Spermine Revealed with a Wider Range of Membrane Voltage
At positive voltages, the spermine-blocking curves in Fig 5 B deviate somewhat from what is predicted by Equation 7, which assumes for simplicity that spermine in the second blocking conformation is strictly nonpermeant. To examine whether the second blocking conformation is also slightly permeant, we examined spermine block over a much wider range of membrane voltage (-180 to +180 mV). We found that, above +90 mV, channel block by spermine was significantly relieved by further depolarization (Fig 6 C), consistent with spermine in the second blocking conformation also being permeant. Interestingly, beyond +140 mV, channel block was again enhanced. These observations argue that spermine blocks the channel in at least three conformations. Thus, to account for this extraordinary voltage dependence of channel block, a third blocking state (c) would need to be added to the model in Fig 9 A. This third conformation (c) should bind to the channel with even less affinity and be less likely to permeate. If the first blocking conformation (a) corresponds to spermine with three (and four) charged amines and the second (b) corresponds to spermine with two charged amines, then the third (c) will correspond to spermine with a single charged amine. This proposal is compatible with the observation that voltage dependence of channel block by the first blocking conformation is stronger than the second, which is stronger than the third, as manifested by the different slopes of the three blocking phases (Fig 6 C). As already mentioned in RESULTS, spermidine appears to behave similarly, although the multiple components are much less well separated (Fig 6 D).
Altering cGMP concentration has different effects on each of the three blocking components of spermine (Fig 7). Lowering cGMP concentration has little effect on the first blocking component, but diminishes the second blocking phase, whereas the third blocking phase essentially vanishes at cGMP concentrations below 30 µM. These results argue that spermine conformation a occupies open and closed channels with similar affinity, b occupies open channels with higher affinity, and c essentially occupies only open channels. Previous studies showed that decreasing cGMP concentrations enhances block of the CNG channel by intracellular tetracaine (
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Footnotes |
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1 Abbreviations used in this paper: ACh, acetylcholine; CNG channel, cyclic nucleotidegated channel; PhTx, philanthotoxin-343.
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Acknowledgements |
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We thank S. Siegelbaum for the CNG channel cDNA clone and R. MacKinnon for the E363G mutant clone, P. De Weer for critical review and discussion of our manuscript, and C.M. Armstrong, S.M. Baylor, and C. Deutsch for helpful discussions.
This study was supported by a National Institutes of Health (NIH) grant (GM55560). Z. Lu was a recipient of an Independent Scientist Award from the NIH (HL03814).
Submitted: 19 August 1999
Revised: 8 May 2000
Accepted: 11 May 2000
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