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* Department of Physiology and Biophysics, University of California, Irvine, California 92697-4561 USA; and Department of Molecular Neurobiology and Cellular Physiology, Institute for Zoology, University of Salzburg, A-5020 Salzburg, Austria
Correspondence: Address reprint requests to Michael D. Cahalan, Dept. of Physiology and Biophysics, University of California, Irvine, CA 92697-4561. Tel.: 949-824-7776; Fax: 949-824-3143; E-mail: mcahalan{at}uci.edu.
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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; Nadler et al., 2001). The human T-cell line, Jurkat, human peripheral T cells, and rat basophilic leukemia (RBL) cells possess a native current with similar properties. Under conditions of divalent-free external solution and Mg2+-free internal solution, a large monovalent current gradually develops during whole cell dialysis in these cells (Kerschbaum and Cahalan, 1998, 1999; Fomina et al., 2000; Braun et al., 2001). The monovalent current consists of the summed activity of tens to hundreds of 
40 pS channels per cell, each opening to a state of a high open probability (Po > 0.9). Using organic monovalent cations of varying size, we estimated the pore diameter of this nonselective cation channel to be 6 Å (Kerschbaum and Cahalan, 1998). Originally, it was proposed that the Ca2+ release-activated Ca2+ (CRAC) channel transforms into a nonselective cation channel upon removal of external divalent cations (Kerschbaum and Cahalan, 1998, 1999). Recently, this view was revised as a result of electrophysiological and molecular biological findings that show differences in the pharmacology, store dependence, and ion selectivity of the divalent and monovalent currents (Hermosura et al. 2002; Prakriya and Lewis, 2002; Kozak et al., 2002). The properties of the native nonselective cation current, named either MagNuM (for magnesium-nucleotide-inhibited metal) (Nadler et al., 2001) or MIC (Mg2+-inhibited cation) (Prakriya and Lewis, 2002) for their inhibition by Mg2+ or Mg2+ nucleotides, match well with cloned and expressed TRPM7 channels (Nadler et al., 2001; Runnels et al., 2001). The most striking electrophysiological similarities between MIC channels and TRPM7 channels are the I/V shape, inhibition by intracellular Mg2+ ion, and permeability to several monovalent and divalent cations (Runnels et al., 2001; Nadler et al., 2001; Prakriya and Lewis, 2002; Kozak et al., 2002). Molecular biological experiments revealed expression of TRPM7 mRNA in Jurkat cells and RBL cells (Nadler et al., 2001).
Although the electrophysiological properties of both MIC and CRAC channels have been studied in some detail, the pharmacology of these channels is still in a premature state. CRAC and MIC are both inhibited by external Ni2+, La3+, and Gd3+ (Runnels et al., 2001; Hermosura et al., 2002). CRAC channels are blocked reversibly by the imidazole antimycotic compound SKF 96365, whereas MIC channels are either insensitive to SKF 96365 in Jurkat cells (Prakriya and Lewis, 2002), or irreversibly inhibited in RBL cells (Kozak et al., 2002). We recently showed that spermine can distinguish MIC from CRAC channels, blocking monovalent MIC current selectively at micromolar concentrations (Kozak et al., 2002).
Blockers have been used frequently, along with the ion selectivity profile and gating characteristics, to establish a biophysical fingerprint for comparing native channels with those in expression systems. For example, a detailed comparison of blocker potencies and other biophysical characteristics led to the identification of Kv1.3 and IKCa1 as the voltage-gated and Ca2+-activated K+ channels, respectively, in human T cells (reviewed in Cahalan et al., 2001). One goal of the present study was to compare the pharmacological profile of native MIC and expressed TRPM7 channels using polyamines and other polyvalent cations as probes. Here, we compare the action of external Mg2+ with several polyamines: putrescine, spermidine, and spermine that are ubiquitous cytoplasmic metabolites; and philanthotoxin-343 (PhTX-343), a synthetic polyamine analog of the toxin present in the venom of the wasp Philanthus triangulum (Eldefrawi et al., 1988). Analysis of the relief of block at negative potentials suggests a permeant block mechanism and provides an independent estimate of the pore diameter for comparison with previous work on bulky permeant ions (Kerschbaum and Cahalan, 1998). Polyamines specifically blocked monovalent current through native MIC channels and expressed TRPM7 channels. We provide an empirical description of ion permeation and block in terms of an Eyring rate theory model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Electrophysiological recordings from Jurkat, RBL, and CHO cells
Macroscopic and single-channel currents were recorded in the whole-cell recording configurations (Hamill et al., 1981) at room temperature using an EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany). Data were acquired and analyzed using Pulse/Pulsefit (v. 8.11) (HEKA), Igor Pro (v. 3.1.2) (WaveMetrics, Lake Oswego, OR), and Microcal Origin (v. 6) (Microcal Software, Northampton, MA) software.
Pipettes were pulled from soft glass capillaries (Becton-Dickinson, Parsippany, NJ), coated with Sylgard (Dow Corning, Midland, MI), and fire-polished to a resistance of 2–5 M
when filled with internal solutions. The glass coverslip chambers used for Jurkat T cell recordings were coated with poly-L-lysine (1 mg/ml) to improve adherence to the dish. Currents were sampled at 5–25 kHz and digitally filtered offline at 1 kHz. The membrane potential was held at 0 mV and currents were studied during 200-msec voltage ramps from -120 mV to +40 mV or during voltage steps from 0 mV to -120 mV. To measure the amplitude of the monovalent current through MIC channels at a given potential more accurately, we applied voltage steps. Voltage ramp or step stimuli were delivered at 1 Hz. Leak currents before activation of MIC channels were averaged and subtracted from subsequent current records. Slow and fast capacitative transients were canceled by the compensation circuitry of the EPC-9. Series resistance (10 M) was not compensated. Quantitative analysis of block was restricted to cells and membrane potentials at which control currents were <0.5 nA and errors due to uncompensated series resistance negligible. Cells were superfused with various solutions by bath exchange. Local solution exchanges were performed via puffer pipettes, as described previously (Lepple-Wienhues and Cahalan, 1996). Durations of open and closed events were estimated from idealized single channel data using TAC software (Bruxton; Seattle, WA). Currents were sampled at a rate of 5–10 kHz and filtered with a Gaussian filter at 1 kHz, resulting in a rise time of 330 µs. Channel opening and closing events were detected using a half-amplitude threshold paradigm, allowing detection of events of 200 µs duration. Data were not corrected for missed events. Idealized current traces were used to generate dwell-time histograms and estimated open and closed durations by fitting the data to a probability density function. At moderate concentrations of polyvalent cations, clear opening and closing events were resolved. Time constants for open and closed times, 
open and closed, respectively, were calculated from probability density functions assuming a single exponential distribution. Channel open probability (Po) was calculated from an all-points amplitude histogram, or from the ratio open/(open + closed); both methods yielded similar values. Calculation of K1/2, the concentration of blocker that reduced current amplitude by half, and the Boltzmann term k representing the steepness of voltage-dependent block, were performed with Igor Pro and Microcal Origin software. Ted Begenisich kindly provided the program that we used to calculate current-voltage relations from a four-barrier, three-site Eyring rate model.
Solutions
Jurkat T lymphocytes
Divalent-free external solution contained (mM): 150 Na+ methane sulfonate or Cs+ methane sulfonate, 10 HEDTA, and 10 HEPES, pH 7.2. MgCl2 was added to the external solution to achieve the desired external free Mg2+ as computed with MaxChelator (Bers et al., 1994). The pipette solution contained (mM): 150 Cs+ aspartate or Na+ aspartate, 10 Cs+-HEPES or Na+-HEPES, 12 BAPTA, and 0.9 CaCl2, pH 7.2 titrated with CsOH or NaOH. All chemicals were purchased from Sigma (St Louis, MO).
RBL and CHO cells
The Ca2+ external solution contained (mM): 2 CaCl2, 167 Na+ aspartate, 2 Cs+ methanesulfonate, and 10 HEPES, pH 7.3 titrated with NaOH. The divalent-free external solution consisted of 154 Cs+ aspartate, 5 NaCl, 10 HEDTA, 2 Cs+ methanesulfonate, and 10 HEPES, pH 7.3 titrated with CsOH. The internal solution contained: 130 Cs+ glutamate, 8 NaCl, 0.9 CaCl2, 12 EGTA, and 10 HEPES, pH 7.3 titrated with CsOH. Spermine hydrochloride (Calbiochem, La Jolla, CA) was added to the divalent-free external solution.
Expression of TRPM7 in CHO-K1 cells
CHO cells were grown in six-well plates and transiently transfected with the mouse TRPM7 clone in the pTracerCMV2 vector using the Effectene transfection kit (Qiagen, Valencia, CA) according to the manufacturer's procedure. The cells were replated on glass coverslips 24 h before electrophysiological recordings. Recordings were made 3–4 days after transfection. The transfected cells were visualized by green fluorescent protein fluorescence. To compare spermine block of the expressed TRPM7 current to the native MIC current in RBL cells, identical ramp protocols were applied (from -120 mV to +85 mV, 211 ms duration, applied at 0.5 Hz). Upon full development of MIC current in Ca2+-containing external solution, the external solution was switched to divalent-free Cs+-HEDTA solution and spermine added.
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RESULTS |
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; Araneda et al., 1999; Guo and Lu, 2000a,b; Huang and Moczydlowski, 2001). Our findings indicate that polyamines are fast blockers that can approach an externally accessible high-affinity binding site within the MIC channel and can pass through the channel driven by the electric field.
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; Prakriya and Lewis, 2002), although this inhibition is not voltage-dependent and may not involve direct interaction with the pore (Kozak et al., 2002). Tested from the inside by inclusion in the pipette solution, 300 µM spermine produced no effect on the development of MIC current or I/V characteristics (n = 10 cells; data not shown). These experiments indicate that spermine, at >1000 times the effective outside dose, cannot readily access the high-affinity binding site from the cytoplasmic side of the plasma membrane and thus suggest an asymmetric pore.
To characterize external polyamine block further, we examined the action of PhTX-343. PhTX-343 blocks several other channel types, but because of its larger size (see structures in Fig. 1), does not exhibit permeant block (punch-through) in the KIR and CNG-gated channels (Guo and Lu, 2000a,b). Extracellular application of PhTX-343 blocked monovalent MIC current in a voltage-dependent manner with relief of block at both depolarized and hyperpolarized potentials (Fig. 3). The potency and voltage dependence is similar to that seen with spermine. A quick way to assess the voltage dependence of block is to divide I/V curves in the presence of blocker by a control I/V curve with no blocker present. As seen in Fig. 3 B, the resultant I/V ratio reflects the fraction of unblocked channels at each potential during a voltage ramp; block by PhTX-343 is most potent at the minimum of this curve from near -50 to -70 mV, and relief of block can be clearly observed at relatively depolarized and hyperpolarized voltages.
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50% and the residual current trace exhibits increased fluctuations noisy during exposure to spermine (Fig. 4 A). During the step to +40 mV, the monovalent outward current was hardly affected by spermine, indicating a rapid relief of block at positive potentials. Finally, upon stepping back to -40 mV, block was immediately restored on a timescale faster than the resolution of the recording (<1 ms). Fast block and unblock kinetics were also observed for spermidine and putrescine (data not shown). In contrast, PhTX-343 block was slower than spermine to equilibrate at -40 mV ( - 11 ms) (Fig. 4 B). We conclude that spermine and related cytoplasmic polyamines exhibit block and unblock kinetics that are faster than the millisecond timescale and cannot be resolved temporally using this step paradigm. The nearly instantaneous block and relief of block justifies the use of voltage ramps for analysis of dose-response relationships. The slower equilibration of block by PhTX-343 can distort the apparent voltage dependence obtained using voltage ramp stimuli.
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90 nM, followed by the trivalent spermidine at two log units lower potency, and then by the divalent putrescine again separated by two log units. As expected from visual inspection of the ramp currents before and after application of the polyamine, the K1/2 value depends strongly on the membrane potential. Fig. 5, B–D, shows experimental data and corresponding Langmuir fits for spermine, spermidine, and putrescine block measured at -120 mV, -40 mV, and +40 mV. K1/2 values were lowest at -40 mV and higher at depolarized or hyperpolarized potentials.
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, the steepness of block depends on the valence (z) of the blocker and the fraction of the electric field (
from the outside) that the blocker has to traverse to reach its binding site. The steepness, expressed as the z product, increased from 1.6 for putrescine (z = 2), to 2.4 for spermidine (z = 3), to 5.2 for spermine (z = 4). For putrescine and spermidine, the result may indicate access from the outside to a binding site most of the way through the membrane, although the situation is likely to be more complex since polyamines have charges distributed along the elongated acyl chain (Fig. 1). Moreover, for spermine (and PhTX-343), the fit to our experimental data would indicate a binding site at an electrical distance larger than unity ( = 1.3, see Discussion). Increasing polyamine concentration did not change the steepness, but shifted V0.5 to the right.
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Because of the voltage dependence of polyamine block, higher concentrations were required before block of outward current was observed. Fig. 7 A shows the effect of spermine at 200 µM, 2 mM, and 20 mM in an RBL cell. At 200 µM, the inward MIC current was completely blocked, whereas 20 mM was required to block most of the outward current. Block of the outward current is accompanied by changes in the shape of the I/V, thus at high spermine concentrations the outward current became slightly concave, similar to the MIC I/V in presence of millimolar external divalents (Kozak et al., 2002). Ca2+ and Mg2+ are permeant ions that can carry current through MIC channels (Nadler et al., 2001; Hermosura et al., 2002; Kozak et al., 2002). We attempted to detect inward current carried by spermine at high concentrations, as would be expected for permeant ion block. Fig. 7 B presents the MIC current I/V curves from panel A on an expanded scale and shows that increasing external spermine concentrations decreased MIC current at both positive and negative potentials; inward current carried by spermine could not be detected. Other investigators have shown that polyamines can act as charge carriers in glutamate channels (Bähring et al., 1997).
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F, where R, T, and F have their usual meanings and represents the equivalent electrical distance across the membrane—indicates a z
product of 1.7 ± 0.4 (n = 3) for Mg2+. Lines fitted to the data are derived from the four-barrier, three-site Eyring rate model described in Discussion.
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). Both Mg2+ and spermine, added to the extracellular solution at 0.3 µM–10 µM, produced frequent interruptions in the unitary inward current that represent block of the single channel; increasing concentrations increased the frequency of these flickery block events. Mg2+ (1 µM) reduced Po from 0.94 ± 0.04 (n = 8 at -120 mV) in the absence of external polyvalent cations to 0.62 ± 0.16 (n = 5); here, changes in Po represent the reduction in current induced by the blocker, not gating transitions. To compare Mg2+ block of macroscopic and single-channel currents, we calculated the apparent dissociation constant from Kd = koff/kon, where koff and kon are rates of unblock and block and correspond to reciprocals of closed and open determined from channel lifetime analysis. The Kd for Mg2+ block from single-channel analysis was 1.9 ± 0.5 µM (n = 5), similar to the K1/2 value of 3 µM estimated from Mg2+ block of the macroscopic current at -120 mV (Fig. 8 B). At 10 µM external Mg2+, individual openings and closings were too fast to resolve clearly. Spermine also blocked single MIC channels (Fig. 9 B), causing Po to decrease to 0.79 ± 0.02 (n = 4) and 0.21 ± 0.04 (n = 5) at 300 nM and 3 µM, respectively. The corresponding calculated Kd values of 1.1 and 0.8 µM agree well with K1/2 of 1 µM determined for spermine block of the macroscopic current (Fig. 5 B). Thus, single-channel analysis reinforces the conclusion that Mg2+ or spermine block the channel by binding to a high-affinity site, reducing macroscopic MIC current by flickery block of the unitary current.
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; Prakriya and Lewis, 2002; Kozak et al., 2002; reviewed in Clapham, 2002; Bakowski and Parekh, 2002). As an additional test, we compared spermine block of TRPM7 current heterologously expressed in CHO cells with native MIC current in RBL cells (Fig. 10, A and B, respectively). Spermine, applied at 200 nM, 2 µM, and 20 µM blocked the TRPM7 monovalent current (in this case carried by Cs+) in a dose- and voltage-dependent manner. Panel B shows the MIC current block by the same concentrations of spermine under identical conditions in an RBL cell. The voltage dependence of spermine block appears to be similar for both TRPM7 and MIC. A more detailed comparison of the blocking effect of spermine is shown in panel C: the I/Vs in the presence of 2 µM spermine obtained from an RBL cell and a CHO cell transfected with TRPM7 are superimposed and scaled. Although the amount of outward current reduction is slightly less in the TRPM7-transfected cell, the shapes of the I/V in the inward direction are identical, showing that the mechanism of block is the same and providing evidence for a nearly identical binding site within the pores of native MIC and expressed TRPM7 channels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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85% down the electric field where it binds tightly enough to block Na+ current. This equivalent electrical distance is similar to that previously determined for Ca2+ ions blocking monovalent current from the outside, indicating a common high-affinity binding site for Mg2+ and Ca2+ (Kerschbaum and Cahalan, 1998).
The externally accessible site for Mg2+ (or Ca2+) is distinct from the internal Mg2+ site that controls current development during whole-cell recording. The channel opens when the cytoplasm is depleted of Mg2+ during whole-cell recording and dialysis. Mg2+ in the high micromolar to low millimolar range blocks without affecting the I/V shape. In cells with preactivated MIC channels, the block by internal Mg2+ is slow to equilibrate, taking longer than expected for Mg2+ to diffuse into the cell. This may indicate that Mg2+ does not exert a direct effect upon the channel by simple block (Kozak et al., 2002). The internal site for Mg2+ block remains uncertain, but is certainly distinct from the externally accessible Mg2+ or polyamine block site.
Block by external polyamines, like that of external Mg2+, is voltage-dependent, indicating external access to a site within the electric field of the membrane (Figs. 2–5). Charges are distributed along the alkyl chain of polyamines, and the sensitivity to membrane potential parallels the number of charges. Table 1 summarizes K1/2 values measured at -40 mV and the steepness factor k, determined by fitting ratio I/V curves. Surprisingly, the longest polyamines tested, spermine and PhTX-343, each blocked with a steepness that would indicate a fractional distance across the electric field, , of >1, similar to steeply voltage-dependent polyamine block reported for inward rectifier potassium channels (Fakler et al., 1995; Lopatin et al., 1994; Lopatin et al., 1995; Pearson and Nichols, 1998). These results point to multiple molecules simultaneously being able to block within the electric field of the channel or to a coupled movement of the blocking ion and the permeant ion through the ion channel (Lopatin et al., 1994, 1995; Fakler et al., 1995; Oliver et al., 1998; Pearson and Nichols, 1998; Oliver et al., 2000). Thus, spermine could interact with its positive charges simultaneously at different binding sites of the MIC channel and sweep out associated permeating monovalent cations.
The relief of Mg2+ block that occurs at very hyperpolarized membrane potentials may be accounted for by "punch-through," in which hyperpolarization provides the sparingly permeant Mg2+ ion with sufficient energy to "knock off" Mg2+ from the binding site, enabling Na+ to carry current again. Block by polyamines can also be relieved by membrane hyperpolarization, consistent with a small but finite permeability through the channel by all compounds studied. Ca2+ and Mg2+ currents through MIC and expressed TRPM7 channels have been directly measured (Nadler et al., 2001; Hermosura et al., 2002; Kozak et al., 2002), and thus it is not surprising that Mg2+ can act as a permeant blocker.
Modeling the block
The simplest model for a permeant blocker represents a binding site within the channel pore that can be occupied by a single permeant ion or by a blocking ion, corresponding to a two-barrier, one-binding site model of the type originally described by Woodhull (1973):
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), with a single high-affinity site for binding within the electric field by polyamines or Mg2+, flanked by two low-affinity sites. The cation pathway through the channel is depicted as a sequential series of four barriers and three wells, allowing multiple occupancy of the channel by more than one ion.
Starting with Na+, we established a set of barriers and wells that gave approximately the right I/V shape and single-channel conductance values (Fig. 11 A, dashed lines). Outer and inner barriers were set to 8.7 RT, corresponding to diffusion-limited access to the pore, and well depths were adjusted empirically to produce the resulting I/V curve in Fig. 11 B. The model produces the characteristic weak inward rectification of MIC current in symmetrical Na+ and zero divalent, with a single-channel current of 4.5 pA at -120 mV, in accord with direct measurement (Kerschbaum and Cahalan, 1999). Next, based on the steepness and potency of block by spermine or Mg2+ at depolarized potentials, we incorporated a high-affinity binding site most of the way toward the inner surface of the membrane. In physical terms, the deep energy well could be provided by fixed negative charges with in the pore, such as glutamate residues that are present in the putative pore region of TRPM7. The well depth was calculated from the apparent dissociation constant by G = -RT ln (Kd), where Kd = to koff/kon. Based on experimental data, we postulated a high-affinity binding site for Mg2+ of -15 RT (which represents a Kd value of 0.8 µM at -40 mV) at a of 0.80. The position and well depth of the high-affinity site and the location of the flanking low-affinity binding sites as well as magnitude of the surface potential were then fine-tuned in an effort to mimic the available experimental data. The final energy profile for Mg2+ is shown by the solid lines in Fig. 11 A. Calculated I/V curves at 3 µM and 3 mM Mg2+ are illustrated in Fig. 11, B and C, respectively. Note that the model provides an accurate fit to the shape of the I/V curve at 3 µM Mg2+, a concentration at which Mg2+ acts as a voltage-dependent blocker whereas Na+ carries the current and also reproduces the characteristic outwardly rectifying MIC I/V shape at 3 mM Mg2+, a concentration at which Mg2+ carries the inward current. At millimolar levels of Mg2+, the small predicted Mg2+ current is 1% of the corresponding predicted Na+ current in the absence of divalent. This predicted Mg2+ current is in accord with a small measured Mg2+ current (Kozak et al., 2002). Finally, the model also provides an excellent fit to I/V ratios at varying concentrations of external Mg2+, as shown in Fig. 8 D, indicating that the model adequately accounts for characteristics of voltage-dependent Mg2+ block and relief of block at more hyperpolarized membrane potentials. At millimolar concentrations, internal Mg2+ inhibits MIC current without altering the shape of the I/V curve (Kozak et al., 2002). Apparently, Mg2+ is unable to access the pore from the inside. For spermine, a well depth of -15 RT and of 0.8 also worked quite well in predicting voltage-dependent block at single concentrations (Fig. 6 A), and predicted block of outward currents in the correct range of concentrations (cf. Figs. 11 D and 7 A), but did not work accurately at spermine concentrations below 1 µM (note deviations in Fig. 6 A). In addition, the model predicts an inward spermine current at high concentrations that could not be detected (Fig. 7 B), and predicts inward rectification induced by internal spermine at concentrations in the high micromolar range, an effect that also was not observed. To summarize, the Eyring rate approach yielded a more accurate fit to data for block by external Mg2+ than for spermine. This difference may be due to the distribution of charge along the spermine molecule that permits spermine to interact simultaneously with more than one site or to interact with permeant ions, features not incorporated in our model. Further studies on ion permeation and block are needed to investigate the asymmetry of the MIC channel.
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). Our data are consistent with previous conclusions that the native MIC current passes through channels composed of TRPM7 subunits.
Comparing permeation and pores of MIC/TRPM7 and other channels
Traditionally, the minimal pore size of an ion channel can be estimated by the largest permeant ion that can carry a measurable current (Hille, 2001). In previous work, we measured the ability of several different organic cations to carry current through the MIC channel. In these studies, MIC current was unmasked by withdrawal of internal Mg2+ and erroneously attributed to the CRAC channel. Nevertheless, the results apply to the MIC channel and provide an estimated pore diameter of 5.8 Å; even tetramethylammonium with a diameter of 5.5 Å is a permeant ion (Kerschbaum and Cahalan, 1998). This procedure for determining the pore diameter excludes sparingly permeant compounds whose current is not resolvable (Burnashev et al., 1996). Alternatively, the minimal pore diameter of ion channels can be estimated from the size of permeant blockers. Spermine, the largest of the three cytoplasmic polyamines tested with a length of 20 Å and a diameter of 4.6 Å, behaved as a permeant blocker and would be able to fit easily through a pore of 6 Å. Surprisingly, even PhTX-343 exhibited punch-through, indicating that the pore can accommodate a molecule of 7.4 Å in diameter. Similar discrepancies between pore size estimated from permeant ions and permeant blockers have been documented for other channel types. Spermine is a permeant blocker of the KIR channel and the voltage-gated Na+ channel with estimated pore sizes of 3 Å and 3 x 5 Å based on the largest permeant ions (Guo and Lu, 2000b; Huang and Moczydlowski, 2001; Hille, 2001). The GluR6 channel is larger; spermine can carry measureable current, and PhTX exerts permeant block (Bähring et al., 1997; Bähring and Mayer, 1998). Several properties of polyamine block in different channel types are compared in Table 2; permeant blockers generally give a larger estimate of the pore size than permeant ions that carry measurable current.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Austrian Science Fund (13395-MOB to H.H.K.) and by National Institutes of Health grant NS14609 (to M.D.C.).
Submitted on September 26, 2002; accepted for publication November 21, 2002.
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REFERENCES |
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Bähring, R., D. Bowie, M. Benveniste, and M. L. Mayer. 1997. Permeation and block of rat GluR6 glutamate receptor channels by internal and external polyamines. J. Physiol. (Lond.). 502:575–589.[Medline]
Bähring, R., and M. L. Mayer. 1998. An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels. J. Physiol. (Lond.). 509:635–650.
Bakowski, D., and A. B. Parekh. 2002. Permeation through store-operated CRAC channels in divalent-free solutions: potential problems and implications for putative CRAC channel genes. Cell Calcium. 32:379–391.[Medline]
Bers, D. M., C. W. Patton, and R. Nuccitelli. 1994. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 40:3–29.[Medline]
Braun, F. J., L. M. Broad, D. L Armstrong, and J. W. Putney Jr. 2001. Stable activation of single Ca2+ release-activated Ca2+ channels in divalent cation-free solutions. J. Biol. Chem. 276:1063–1070.
Burnashev, N., A. Villarroel, and B. Sakmann. 1996. Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues. J. Physiol. (Lond.). 496:165–173.[Medline]
Cahalan, M. D., H. Wulff, and K. G. Chandy. 2001. Molecular properties and physiological roles of ion channels in T lymphocytes. J. Clin. Immunol. 21:235–252.[Medline]
Clapham, D. E. 2002. Sorting out MIC, TRP, and CRAC Ion Channels. J. Gen. Physiol. 120:217–220.
Eldefrawi, A. T., M. E. Eldefrawi, K. Konno, N. A. Mansour, K. Nakanishi, E. Oltz, and P. N. Usherwood. 1988. Structure and synthesis of a potent glutamate receptor antagonist in wasp venom. Proc. Natl. Acad. Sci. USA. 85:4910–4913.
Fakler, B., U. Brandle, E. Glowatzki, S. Weidemann, H. P. Zenner, and J. P. Ruppersberg. 1995. Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine. Cell. 80:149–154.[Medline]
Fomina, A. F., C. M. Fanger, J. A. Kozak, and M D. Cahalan. 2000. Single channel properties and regulated expression of Ca2+ release-activated Ca2+ (CRAC) channels in human T cells. J. Cell. Biol. 150:1435–1444.
Guo, D., and Z. Lu. 2000a. Mechanism of cGMP-gated channel block by intracellular polyamines. J. Gen. Physiol. 115:783–798.
Guo, D., and Z. Lu. 2000b. Mechanism of IRK1 channel block by intracellular polyamines. J. Gen. Physiol. 115:799–814.
Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391:85–100.[Medline]
Hermosura, M. C., M. K. Monteilh-Zoller, A. M. Scharenberg, R. Penner, and A. Fleig. 2002. Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNuM. J. Physiol. (Lond.). 539:445–458.
Hille, B. 2001. Ion Channels of Excitable Membranes, 3rd ed. Sinauer Associates, Sunderland, MA.
Huang, C. J., and E. Moczydlowski. 2001. Cytoplasmic polyamines as permeant blockers and modulators of the voltage-gated sodium channel. Biophys. J. 80:1262–1279.
Kerschbaum, H. H., and M. D. Cahalan. 1998. Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes. J. Gen. Physiol. 111:521–537.
Kerschbaum, H. H., and M. D. Cahalan. 1999. Single channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes. Science. 283:836–839.
Kozak, J. A., H. H. Kerschbaum, and M. D. Cahalan. 2002. Distinct properties of CRAC and MIC channels in RBL cells. J. Gen. Physiol. 120:221–235.
Lepple-Wienhues, A., and M. D. Cahalan. 1996. Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells. Biophys. J. 71:787–794.
Lopatin, A. N., E. N. Makhina, and C. G. Nichols. 1994. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature. 372:366–369.[Medline]
Lopatin, A. N., E. N. Makhina, and C. G. Nichols. 1995. The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines. J. Gen. Physiol. 106:923–955.
Lu, Z., and L. Ding. 1999. Blockade of a retinal cGMP-gated channel by polyamines. J. Gen. Physiol. 113:35–43.
McCleskey, E. 1999. Calcium channel permeation: a field in flux. J. Gen. Physiol. 113:765–772.
Nadler, M. J., M. C. Hermosura, K. Inabe, A. L. Perraud, Q. Zhu, A. J. Stokes, T. Kurosaki, J. P. Kinet, R. Penner, A. M. Scharenberg, and A. Fleig. 2001. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature. 411:590–595.[Medline]
Oliver, D., H. Hahn, C. Antz, J. P. Ruppersberg, and B. Fakler. 1998. Interaction of permeant and blocking ions in cloned inward-rectifier K+ channels. Biophys. J. 74:2318–2326.
Oliver, D., T. Baukrowitz, and B. Fakler. 2000. Polyamines as gating molecules of inward-rectifier K+ channels. Eur. J. Biochem. 267:5824–5829.[Medline]
Pearson, W. L., and C. G. Nichols. 1998. Block of the Kir2.1 channel pore by alkylamine analogues of endogenous polyamines. J. Gen. Physiol. 112:351–363.
Prakriya, M., and R. S. Lewis. 2002. Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels. J. Gen. Physiol. 119:487–507.
Runnels, L. W., L. Yue, and D. E. Clapham. 2001. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science. 291:1043–1047.
Wollmuth, L. P., T. Kuner, P. H. Seeburg, and B. Sakmann. 1996. Differential contribution of the NR1- and NR2A-subunits to the selectivity filter of recombinant NMDA receptor channels. J. Physiol. (Lond.). 491:779–797.[Medline]
Woodhull, A. M. 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61:687–708.
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