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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Intracellular polyamines inhibit the strongly rectifying IRK1 potassium channel by a mechanism different from that of a typical ionic pore blocker such as tetraethylammonium. As in other K+ channels, in the presence of intracellular TEA, the IRK1 channel current decreases with increasing membrane voltage and eventually approaches zero. However, in the presence of intracellular polyamines, the channel current varies with membrane voltage in a complex manner: when membrane voltage is increased, the current decreases in two phases separated by a hump. Furthermore, contrary to the expectation for a nonpermeant ionic pore blocker, a significant residual IRK1 current persists at very positive membrane voltages; the amplitude of the residual current decreases with increasing polyamine concentration. This complex blocking behavior of polyamines can be accounted for by a minimal model whereby intracellular polyamines inhibit the IRK1 channel by inducing two blocked channel states. In each of the blocked states, a polyamine is bound with characteristic affinity and probability of traversing the pore. The proposal that polyamines traverse the pore at finite rates is supported by the observation that philanthotoxin-343 (spermine with a bulky chemical group attached to one end) acts as a nonpermeant ionic blocker in the IRK1 channel.
Key Words: inward-rectifier K+ channel, ion permeation, protonation, polyamine, diamine
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INTRODUCTION |
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Inward-rectifier K+ channels are blocked by intracellular Mg2+ and polyamines (
Previous studies of polyamine block of strongly rectifying inward-rectifier K+ channels have revealed complex phenomena. For example, the relation between the extent of channel block and membrane voltage exhibits a shallower phase and a steep phase, and thus requires an equation with two Boltzmann terms (
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METHODS |
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Molecular Biology and Oocyte Preparation
IRK1 (Kir2.1) cDNA cloned into the pcDNA1/AMP plasmid (Invitrogen Corp.) was kindly provided by Dr. Lily Y. Jan (
Patch Recording and Data Analysis
IRK1 currents were recorded in the inside-out configuration from the membrane of Xenopus oocytes (injected with IRK1 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. During the current recording, the voltage across the membrane patch was first hyperpolarized from the 0 mV holding potential to -100 mV for 50 ms (to relieve channel block by the tested intracellular blocker), and then stepped to a test voltage between -100 and +100 mV, in 5- or 10-mV increments, for 0.31 s. Background leak current correction was carried out as previously described (
Recording Solutions
Pipette solution contained (mM): 100 KCl, 0.3 CaCl2, 1.0 MgCl2, and 10 HEPES, pH 7.6. The bath solutions contained (mM): 90 KCl, 5 K2EDTA, 10 HEPES, pH 7.6 or 6.6, as specified. The bath solutions containing diamines, polyamines, and philanthotoxin-343 (PhTx)1 were prepared daily.
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RESULTS |
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Channel Block by Intracellular Tetraethylammonium
Tetraethylammonium and all other intracellular IRK1 channel blockers that we studied block the channel in a voltage-dependent manner. To examine the IRK1 current at various membrane voltages, we used a voltage-pulse protocol rather than a ramp, because the current through the IRK1 channel does not instantaneously reach a steady state after a change in membrane voltage (Fig 1 A). To quantify channel block by a given blocker, we analyzed its effect only on the steady state current. Although the nature of the time-dependent current decline after a step to positive membrane voltages is unknown, intrinsic gating has been suggested as the cause because the current decline persists even after the intracellular side of the membrane patch is exhaustively perfused with recording solution nominally devoid of blocking ions (
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For the purpose of comparison, we first examined channel block by the classic intracellular ionic pore blocker TEA (
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Channel Block by Intracellular Putrescine
Fig 3 A shows current traces recorded in the absence or presence of various concentrations of intracellular putrescine. The recordings were carried out between -100 and +100 mV in 5-mV increments, but, for clarity, we plotted only the current traces recorded at 10-mV intervals. Fig 3 B plots the I-V curves in the absence and presence of the three concentrations of putrescine. Unlike that in the presence of TEA (compare Fig 2 B), the IRK1 channel current in the presence of putrescine, after reaching a peak at some positive voltages, did not continue to decline towards zero in the manner expected for a typical ionic pore blocker. Rather, a significant current persists at very positive membrane voltages. The amplitude of the residual current is smaller at higher putrescine concentrations. In Fig 3 C, we plotted the fractions of unblocked current as a function of membrane voltage. Contrary to the expectation for voltage-dependent block by a typical ionic pore blocker, the extent of channel block for a given putrescine concentration does not follow the Woodhull equation, but approaches a nonzero level at positive membrane voltages. Putrescine clearly does not simply act as a typical ionic pore blocker.
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Channel Block by Intracellular Diamines
To better understand block of the IRK1 channel by putrescine (a primary diamine) described here, we systematically tested a series of primary diamines of varying methylene chain length. Fig 4 shows the current traces of the IRK1 channel between -100 and +100 mV, recorded in the absence and presence of nine diamines containing from 2 to 10 methylene groups (DMC2 through DMC10; putrescine is DMC4). Fig 5 shows I-V curves obtained without and with the nine diamines at various concentrations including those of Fig 4. Except for DMC2, which blocked the channel slightly more effectively than DMC3, blocking efficacy generally increased with methylene chain length. The effect of each diamine on the I-V curves is qualitatively similar to that of putrescine (DMC4). However, the currents appear to reach a nonzero "plateau" at progressively lower membrane voltages as the methylene chain length increases. In Fig 6, we plotted the fractions of unblocked current in the presence of the various diamines as a function of membrane voltage. As in the case of putrescine (DMC4), the extent of channel block by each of the diamines tends to a nonzero level at positive membrane voltages, a behavior reminiscent of block of the retinal cGMP-gated (CNG) channel by certain diamines (e.g., DMC8, DMC9, and DMC10), where these diamines act as permeant blockers (
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Channel Block by Intracellular Polyamines
Fig 7 A shows the current traces recorded between -100 and +100 mV in the absence or presence of various concentrations of the triamine spermidine. Practically, 30 nM is the lowest concentration of spermidine at which the outward IRK1 current reaches a steady state within 300 ms after a voltage step. In Fig 7 B, we plotted I-V curves recorded in the absence or presence of spermidine at three concentrations. The I-V curve in the presence of spermidine consists of multiple phases. Fig 7 C shows the fractions of unblocked current at the three spermidine concentrations as a function of membrane voltage. As with diamines, the extent of channel block by spermidine tends to nonzero levels at positive membrane voltages, but a noticeable hump appears in spermidine-blocking curves that was absent from those of diamines.
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Fig 8 A shows channel block by the intracellular tetramine spermine. Although spermine appears to block the channel more effectively, it displays a complex blocking behavior generally similar to that of spermidine. The I-V curves in the presence of spermine also consist of multiple phases (Fig 8 B). The spermine-blocking curves also have a hump, albeit less prominent than in the case of spermidine, preceding the plateau phase at positive voltages (Fig 8 C). Over a comparable voltage range, the IRK1 channel blocking curves for spermine and spermidine shown here resemble somewhat those in the CNG channel (
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Channel Block by Spermidine at Two Different Intracellular pH
To test whether the multiphasic polyamine blocking curve is due to nonuniform protonation of the polyamine and/or the channel, we examined how intracellular pH affects channel block by spermidine. Fig 9 A shows IRK1 channel current traces obtained at membrane voltages between -100 and +100 mV. The two sets of current traces Fig 9 A, top, were recorded without and those below with intracellular spermidine. Fig 9 A, left, were obtained at intracellular pH 7.6, those on the right at pH 6.6. As shown previously (
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Channel Block by a Spermine Derivative
To test whether the residual current at positive voltages is due to spermine being a permeant blocker, we examined block of the IRK1 channel by PhTx, essentially spermine with a bulky chemical group attached to one end. Fig 10 A shows the current traces recorded at membrane voltages between -100 and +100 mV in the absence or presence of 10 µM PhTx. The corresponding I-V curves are shown in Fig 10 B. In Fig 10 C, we plotted the fractions of unblocked current as a function of membrane voltage. As in the case of TEA, the extent of channel block by PhTx increased monotonically with membrane voltage. Both the hump and the trailing "plateau" phase seen in the spermine-blocking curve are absent. The curve superimposed on the data points in Fig 10 C is a fit of the Woodhull equation. Thus, PhTx blocks the IRK1 channel very much like a typical (nonpermeant) ionic pore blocker.
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DISCUSSION |
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Block of the IRK1 channel by intracellular polyamines appears to be complex. To understand its mechanism, we examined channel blocking behaviors of di- and polyamines as a function of concentration and membrane voltage. Some of these blocking behaviors are reminiscent of those occurring in the CNG channel (
Channel Block by Diamines
As observed previously by
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(1) |
or
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(2) |
where [DM] is the intracellular diamine concentration (the extracellular diamine concentration is zero) and all rate constants are assumed to vary exponentially with membrane voltage. The four independent parameters are: K1, the equilibrium dissociation constant for channel-diamine interaction; k-2/k-1, the relative probability of a diamine bound in the pore permeating the pore versus returning to the intracellular solution; and the corresponding apparent valences Z1 and "z-1 + z-2". The curves superimposed on the data in Fig 6 are fits of Equation 2.
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Values for these four adjustable parameters, determined from the fits, are plotted against diamine methylene chain length in Fig 12. The value of K1 generally decreases with increasing methylene chain length (Fig 12 A), consistent with the observation that longer diamines are more potent blockers (Fig 5). Thus, as previously suggested (
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Fig 12 C shows that k-2/k-1 is rather small and decreases (from 0.05 to 0.01) with increasing methylene chain length. Since k-2/k-1 is the relative probability of a molecule bound in the pore traversing the pore versus returning to the intracellular solution, the result shows that diamines traverse the pore very infrequently. However, a few percent of bound diamine molecules popping through the pore allow sufficient K+ permeation to account for the residual current at positive membrane voltages. Furthermore, k-2/k-1 decreases with increasing methylene chain length, consistent with the expectation that more hydrophobic diamines are less likely to traverse the pore.
The valence factors Z1 and "z-1 + z-2" also increase with methylene chain length (Fig 12B and Fig D). Empirically, Z1 and "z-1 + z-2" for a given diamine appear to be numerically similar. If, for the sake of discussion, one assumes that for a given diamine, quantities Z1 and "z-1 + z-2" are equal, then Equation 1 simplifies to:
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(3) |
At a sufficiently high membrane voltage, Equation 3 approaches a voltage-independent constant value determined solely by diamine concentration:
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(4) |
which explains why at a given diamine concentration the voltage-dependent inhibition curve can be well fitted using the Woodhull equation, provided an extra constant is included to represent residual current at positive membrane voltages (
Equation 4 can be appreciated in an intuitive way. When a lodged diamine molecule pops through the pore, a brief window of opportunity is created for K+ ions to "leak" through the pore; the window closes when another diamine molecule becomes lodged in the pore. Either a higher diamine binding rate (k1[DM]) or a lower rate of bound diamine popping through the pore (k-2), or both, will reduce the time available for K+ to leak and thus leave a smaller residual current. This explains why the residual current is inversely related to diamine concentration (Fig 6). Although diamines may permeate the channel, their intrinsic rate of permeation is too slow to account for the residual current. Nearly all the observed residual current must be carried by K+ ions because it vanishes at sufficiently high blocker concentrations (Fig 6).
Channel Block by Polyamines
Fig 7 C shows the voltage-dependent blocking curve of the IRK1 channel in the presence of three concentrations of the triamine spermidine. Above 0 mV, the curve descends rapidly as membrane voltage is increased, and it then forms a hump before approaching a nonzero level at extreme positive voltages. Clearly, the curve cannot be fitted by a Woodhull equation. These blocking curves are reminiscent of those of the CNG channel in the presence of intracellular polyamines, although the hump observed here is much less prominent (
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(5) |
or, upon rearranging:
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(6) |
where [PM] is the intracellular polyamine concentration (the extracellular polyamine concentration is zero) and all rate constants are assumed to vary exponentially with membrane voltage. As shown in Fig 7, the spermidine-blocking curves can be well fitted by Equation 6.
Since the IRK1 channel is inhibited by intracellular protons, and polyamines are not fully protonated near neutral pH (
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(7) |
where = 10-pH/(10-pH + 10-pKa). The smooth traces superimposed on the blocking curves in Fig 9 B are simultaneous fits of Equation 7 to the two curves. From the fit, we deduced a pKa for the titrated site of 8.1 ± 0.1 (mean ± SEM). Given this value, much larger changes in the blocking curve should be observed if the intracellular pH were raised from 7.6 rather than lowered. Unfortunately, channel currents run down very rapidly above pH 8.0.
Intracellular protonation inhibits the IRK1 channel and also affects its gating, presumably by titrating a site in the channel with apparent pKa = 6.2 (
As shown in Fig 8 C, the blocking behavior of the tetramine spermine is very similar to that of the triamine spermidine and can be accounted for by the same model. However, the blocking behavior of a spermine derivative, PhTx, is quite differentno significant residual current was observed at positive voltages when the channel was blocked by PhTx (Fig 10), as in the glutamate- and cGMP-gated channels (
As in the case of diamines, comparable Z1 and "z-1 + z-2" values for each state (a and b) are needed to fit the polyamine-blocking curves. For the purpose of illustration, if one assumes Za1 and "za-1 + za-2" to be numerically equal (denoted Za) and Zb1 and "zb-1 + zb-2" to be numerically equal (denoted Zb), then Equation 6 becomes Equation 8:
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(8) |
or, upon rearranging:
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(9) |
Furthermore, for spermidine concentrations much greater than the sum of ka-2/ka1 and kb-2/kb1, Equation 9 approaches Equation 10:
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(10) |
showing that the voltage dependence of channel block by polyamines at high concentrations can be approximated by an equation with two Boltzmann terms (compare
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(11) |
producing a hump (Fig 7 C). At extreme positive voltages, Equation 11 approaches Equation 12:
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(12) |
accounting for the residual current at these voltages. As in the case of diamines, the amplitude of the residual current should decrease with increasing spermidine concentration and tend towards zero at very high concentrations, as observed (Fig 7 C).
Currents through the IRK1 channel after a jump to positive membrane voltages decline with time even in patches exhaustively perfused with nominally blocking ion-free solution. It has been suggested that this decline in current results from intrinsic gating (
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(13) |
where K* is [Ch*]/[Ch], and Z* is the associated valence factor. Under our experimental conditions, the fitted values for both K* (0.03 ± 0.01; mean ± SEM) and Z* (0.55 ± 0.01) are small (Fig 1 C), yielding multiplier values that range from 0.97 at 0 mV to 0.80 at +100 mV. Therefore, the addition of this nonconducting state would cause only minor changes in the fitted equilibrium constants (K1a and K1b) and valence factors (Z1a and Z1b), but has no fundamental impact on our analysis.
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Models involving channel gating have previously been proposed to explain various aspects of intracellular cation-induced inward rectification in the IRK1 channel. For example,
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Footnotes |
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1 Abbreviations used in this paper: CNG channel, cyclic nucleotidegated channel; PhTx, philanthotoxin-343.
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Acknowledgements |
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We thank L.Y. Jan for the IRK1 channel cDNA clone, P. De Weer for critical review and discussion of our manuscript, and C.M. Armstrong, C. Deutsch, and G. Yellen for helpful discussions.
This study was supported by National Institutes of Health (NIH) grant GM55560. Z. Lu was a recipient of an Independent Scientist Award from NIH (HL03814).
Submitted: 19 August 1999
Revised: 8 May 2000
Accepted: 11 May 2000
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References |
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Armstrong, C.M., Binstock, L. 1965. Anomalous rectification in the squid giant axon injected with tetraethylammonium. J. Gen. Physiol. 48:859-872
Bähring, R., Bowie, D., Benveniste, M., Mayer, M.L. 1997. Permeation and block of the rat GluR6 glutamate receptor channels by internal and external polyamines. J. Physiol. 502:575-589[Abstract].
Fakler, B., Brandle, U., Glowatzki, E., Weidemann, S., Zenner, H.P., Ruppersburg, J.P. 1995. Strong voltage-dependent inward-rectification of inward-rectifier K+ channels is caused by intracellular spermine. Cell. 80:149-154[Medline].
Ficker, E., Taglialatela, M., Wible, B.A., Henley, C.M., Brown, A.M. 1994. Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science. 266:1068-1072[Medline].
Guo, D., Lu, Z. 2000. Mechanism of cGMP-gated channel block by intracellular polyamines. J. Gen. Physiol. 115:783-797
Hille, B. 1992. Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates, Inc, pp. 607.
Horie, M., Irisawa, H., Noma, H. 1987. Voltage-dependent magnesium block of adenosine- triphosphatesensitive potassium channel in guinea-pig ventricular cells. J. Physiol. 387:251-272[Abstract].
Kubo, Y., Baldwin, T.J., Jan, Y.N., Jan, L.Y. 1993. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature. 362:127-133[Medline].
Lee, J.-K., Scott, J.A., Weiss, J.N. 1999. Novel gating mechanism of polyamine block in the strong inward rectifier K channel Kir2.1. J. Gen. Physiol. 113:555-563
Logothetis, D.E., Kurachi, Y., Galper, J., Neer, E.J., Clapham, D.E. 1987. The ß subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 325:321-326[Medline].
Lopatin, A.N., Makhina, E.N., Nichols, C.G. 1994. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature. 372:366-369[Medline].
Lopatin, A.N., Makhina, E.N., Nichols, C.G. 1995. The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines. J. Gen. Physiol 106:923-955[Abstract].
Lu, T., Nguyen, B., Zhang, X., Yang, J. 1999. Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification. Neuron. 22:571-580[Medline].
Lu, Z., MacKinnon, R. 1994. Electrostatic tuning of Mg2+ affinity in an inward-rectifier K+ channel. Nature. 371:243-246[Medline].
Matsuda, H., Saigusa, A., Irisawa, H. 1987. Ohmic conductance through the inward-rectifier K+ channel and blocking by internal Mg2+. Nature. 325:156-159[Medline].
Oliver, D., Hahn, H., Antz, C., Ruppersberg, J.P., Fakler, B. 1998. Interaction of permeant and blocking ions in cloned inward-rectifier K+ channels. Biophys. J. 74:2318-2326
Pearson, W.L., Nichols, C.G. 1998. Block of the Kir2.1 channel pore by alkylamine analogues of endogenous polyamines. J. Gen. Physiol. 112:351-363
Palmer, B.N., Powell, H.K.J. 1974. Polyamine complex with seven-membered chelate rings: complex formation of 3-azaheptane-1,7-diamine, 4-azaoctane-1,8-diamine (spermidine), and 4,9- diazadodecane-1,12-diamine (spermine) with copper (II) and hydrogen ions in aqueous solution. J. Chem. Soc. Dalton Trans. 19:2089-2092.
Shieh, R.C., John, S.A., Lee, J.-K., Weiss, J.N. 1996. Inward rectification of IRK1 expressed in Xenopus oocytes: effects of intracellular pH reveal an intrinsic gating mechanism. J. Physiol. 494:363-376[Abstract].
Spassova, M., Lu, Z. 1998. Coupled ion movement underlies rectification in an inward-rectifier K+ channel. J. Gen. Physiol. 112:211-221
Spassova, M., Lu, Z. 1999. Tuning the voltage dependence of tetraethylammonium block with permeant ions in an inward-rectifier K+ channel. J. Gen. Physiol. 114:415-426
Vandenberg, C.A. 1987. Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions. Proc. Natl. Acad. Sci. USA. 84:2560-2564[Abstract].
Woodhull, A.M. 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61:687-708
Yang, J., Jan, Y.N., Jan, L.Y. 1995. Control of rectification and permeation by residues in two distinct domains in an inward rectifier K+ channel. Neuron. 14:1047-1054[Medline].