Correspondence to: William A. Sather, Neuroscience Center, B-138, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. Fax:303-315-2503 E-mail:william.sather{at}uchsc.edu.
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Abstract |
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Selective permeability in voltage-gated Ca2+ channels is dependent upon a quartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca2+ binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca2+ binding sites. The current view is based on site-directed mutagenesis work in which Ca2+ binding affinity was attenuated by single and double substitutions in the EEEE locus, and eliminated by quadruple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. However, interpretation of the mutagenesis work can be criticized on the grounds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second site may have remained undetected because the mutated pore was probed only from the extracellular pore entrance. Here, we describe the results of experiments designed to test the strength of these criticisms of the single high-affinity locus model of selective permeability in Ca2+ channels. First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed. Second, the postulated second high-affinity site was not detected by probing from the intracellularly oriented pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we found that, whereas micromolar Ca2+ produced substantial block of outward Li+ current in wild-type channels, internal Ca2+ concentrations up to 1 mM did not produce detectable block of outward Li+ current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca2+ binding locus in the pore of voltage-gated Ca2+ channels.
Key Words: ion selectivity, selectivity filter, L-type Ca2+ channel, patch clamp, cysteine mutagenesis
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INTRODUCTION |
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For voltage-gated Ca2+ channels, selective permeability is believed to involve interaction between two or more permeant ions. However, the number of pore-localized ion binding sites that may mediate these interactions has been contentious, with assertions of two or more binding sites (7.5 Å. This small separation very likely allows significant ionion interaction to occur within the K+ channel's pore, as has also been postulated for Ca2+ channels. In the case of Ca2+ channels, tight binding of one Ca2+ ion within the single-file region of the pore obstructs permeation by foreign ions (selectivity), whereas Ca2+Ca2+ interaction inside the pore overcomes tight Ca2+ binding to generate high Ca2+ throughput (unitary Ca2+ current). These two processes can be observed in an experimental setting, where the probability of pore occupancy by Ca2+ can be manipulated: with approximately micromolar Ca2+ in the bath, the probability of single occupancy by Ca2+ is high, so that Ca2+ effectively blocks monovalent metal cation (e.g., Na+) flux; with approximately millimolar Ca2+ in the bath, the probability of double occupancy by Ca2+ is high, so that Ca2+Ca2+ interactions within the pore are frequent and high unitary Ca2+ flux is achieved (
Site-directed mutagenesis studies have identified a set of four conserved glutamate residues that contribute to Ca2+ binding, with the glutamates referred to in ensemble as the EEEE locus (
Yet the existence of a second high-affinity, pore-localized Ca2+ binding site has remained a lurking possibility, according to either of the following two lines of reasoning. One criticism holds that the single and double mutations introduced into the EEEE locus not only reduced ion binding affinity in that locus, but that, in addition, the mutations disrupted function of the second high-affinity binding site. Such an effect could be viewed as arising from a more extensive disruption of pore structure than is hoped for with site-directed mutagenesis, or more elaborately, as a consequence of disruption by the mutations of a hypothesized allosteric interaction between the two high-affinity sites. A second criticism focuses on the fact that the conclusions of
Irrespective of the specific formulations of these two criticisms, their salience lies in the exposure of a weakness in the case for a single high-affinity locus model of selective permeability in Ca2+ channels. In this paper, we present two tests of the validity of these arguments: we have used the substituted-cysteine accessibility method (
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MATERIALS AND METHODS |
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Molecular Biology and Expression of Ca2+ Channels
To enhance channel expression in Xenopus oocytes, cDNAs for the pore-forming 1C (
2
(
1C cDNA was also reconstructed to include a consensus Kozak sequence for initiation of translation. Quadruple alanine (AAAA) and quadruple glutamine (QQQQ) mutants were made by substituting either alanine (A) or glutamine (Q) for the EEEE locus glutamate (E) in each of the four motifs of wild-type (WT)1
1C. All mutants were constructed using megaprimer PCR-based mutagenesis (
Ovarian tissue was removed from anesthetized female Xenopus laevis (Xenopus One) and agitated in Ca2+-free OR-2 solution (mM: 82.5 NaCl, 2.5 KCl, 1 MgCl2, 5 HEPES, pH 7.6 with NaOH) containing 2 mg/ml collagenase A or B (Boehringer) for 60120 min. After a 30-min rinse in fresh OR-2 solution, stage V and VI oocytes were selected by hand. cRNAs encoding 1C (WT or mutant),
2
, and ß2b subunits were transcribed in vitro using the mMESSAGE mMACHINE kit (Ambion) and injected into oocytes in an equimolar ratio. Injected oocytes were stored at 18°C in ND-96 solution (mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.6 with NaOH) containing 2.5 mM sodium pyruvate (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich), 0.1 mg/ml streptomycin (Sigma-Aldrich), and 0.1 mg/ml gentamicin (Boehringer) and studied 314 d after injection.
For expression in HEK293 cells, WT and mutant cDNAs were subcloned into the pBK-CMV N/B-200 vector, a version of pBK-CMV (Stratagene) in which the lac promoter and the initiation codon for ß-galactosidase have been removed. HEK293 cells (CRL-1573; American Type Culture Collection) were grown at 37°C and 5% CO2 in Minimal Essential Medium (GIBCO BRL) supplemented with 10% fetal bovine serum (GIBCO BRL), 100 U/ml penicillin (Sigma-Aldrich), and 0.1 mg/ml streptomycin (Sigma-Aldrich). An equimolar ratio of 1C,
2
, ß2b, and green fluorescent protein (GFP; GIBCO BRL) cDNAs were transiently transfected using the Effectene transfection kit (QIAGEN). Cells expressing GFP (as determined by their fluorescence) were studied 4872 h after transfection. Approximately 1020% of cells were transfected (expressed GFP) under these conditions.
Electrophysiological Recording from Xenopus Oocytes
Single-channel currents were recorded from cell-attached patches on oocytes that had been stripped of their vitelline membrane. The oocytes were bathed in a high K+ solution designed to zero the membrane potential (mM: 100 KCl, 10 HEPES, 10 EGTA, pH 7.4 with TEA-OH). The Ca2+ channel agonist FPL 64176 (RBI) was included in the bath solution (2 µM) to prolong channel open time, which facilitated the study of pore block. Patch pipets were made of borosilicate glass (Warner Instruments Co.), coated with either Sylgard (Dow Corning Corp.) or wax (Kerr Corp.) and had resistances of 2030 M
when filled with solution for recording inward Li+ currents (mM: 100 LiCl, 10 HEPES, 10 HEDTA, 14 TEA-Cl, plus various concentrations of CaCl2, pH 7.4 with TEA-OH). "Chelator" software (Theo J.M. Schoenmakers, University of Nijmegen, Nijmegen, The Netherlands) was used to determine the [CaCl2] to add to the recording solution for a desired final free [Ca2+]. Currents were recorded with an Axopatch 200B amplifier (Axon Instruments, Inc.). The amplifier's internal filter was set to 10 kHz and an external filter (eight-pole Bessel filter; Frequency Devices, Inc.) was set to 2 kHz, yielding a final corner frequency of 1.96 kHz. Data were sampled at 10 kHz using Pulse software (HEKA, distributed by Instrutech Corp.).
Two-electrode voltage clamp recording from oocytes was performed as previously described (. The bath was continuously perfused (
1 ml/min) with either Li+ solution (same as above for single-channel recording) or Ba2+ solution [mM: 40 Ba(OH)2, 52 TEA-OH, 5 HEPES, pH 7.4 with methane sulfonic acid]. In experiments where amino acid side-chain accessibility was studied in cysteine mutants, the sulfhydryl-modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA·Br; Toronto Research Chemicals) was dissolved in Li+ solution immediately before application to the oocyte via the bath perfusion system. Currents were recorded with an amplifier (OC-725C; Warner Instruments Co.), filtered at 500 Hz (four-pole Bessel filter; Warner Instruments Co.) and sampled at 1 kHz. Computer programs to control the data acquisition and analyze the data were custom written in Axobasic (Axon Instruments, Inc.). Leak currents were subtracted using a modified P/4 protocol: 10 pulses to -P/4 were averaged.
Electrophysiological Recording from HEK293 Cells
Whole-cell currents were recorded from HEK293 cells with a 100 mM Li+ solution (as above for oocytes) in the bath and a Cs+ solution in the pipet (mM: 135 CsCl, 10 EGTA, 10 HEPES, 4 Mg-ATP, pH 7.5 with TEA-OH). Patch pipets were made from thin-walled borosilicate glass (Warner Instruments Co.) and had resistances of 13 M
. Currents were recorded and filtered with the same equipment, settings, and software as described above for single-channel current recording from oocytes, and they were sampled at 4 kHz. Leak currents were subtracted using the average of eight pulses to -P/10. Uncompensated series resistance was <9 M
, and this was compensated by 7080% during experiments.
Following previous work by
All chemicals for which a source is not specifically mentioned were obtained from either Sigma-Aldrich or Fluka. All mean values are reported as mean ± SEM.
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RESULTS |
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Accessibility of Substituted Cysteines Suggests that AAAA Pore Structure Is Not Globally Disrupted
The fact that divalent cations do not permeate the AAAA channel raises the possibility that quadruple mutations in the EEEE locus produce a nonspecific disruption of pore structure, so that divalent cations do not permeate, although monovalent cations do. It has been suggested that this kind of disruption of structure might also destroy the postulated second high-affinity Ca2+ binding site. As a means of assessing the structural integrity of the AAAA channel, we have compared the orientations of amino acid side-chains of pore-lining residues in WT channels with those of the corresponding residues in AAAA channels. Side-chain orientation, that is, whether a side-chain projects into the pore or not, was investigated by measuring whether a bath-applied sulfhydryl-modifying reagent could irreversibly block current through cysteine-substituted EEEE or AAAA channels. We analyzed side-chain orientation at two positions in each motif, the position occupied in WT channels by an EEEE locus glutamate (position 0), and the immediate amino-terminal neighbor (position -1), which is located farther from the channel's external entrance than is the EEEE locus glutamate. For EEEE channels, four 0-position cysteine substitution mutants were studied (CEEE, ECEE, EECE, and EEEC), and, for AAAA channels, four additional 0-position mutants were studied (CAAA, ACAA, AACA, and AAAC). Cysteine substitution at the -1 position yielded four mutants in the EEEE channel (M392C, G735C, F1144C, and G1445C), and four mutants in the AAAA channel (M392C/AAAA, G735C/AAAA, F1144C/AAAA, and G1445C/AAAA).
The effects of the methanethiosulfonate reagent MTSEA on inward, whole-cell Li+ currents carried by non- and cysteine-substituted AAAA channels (0 position only) are illustrated in Fig 1 (A and B). For the noncysteine-substituted AAAA channel, MTSEA block was small and rapidly reversible. The average steady state block of AAAA channels by MTSEA was 18 ± 1% (Fig 2 A), and the time constant for recovery from MTSEA block was 21 ± 6 s (n = 6). This unblock rate is similar to that measured for Cd2+ unblock in oocytes, and most likely reflects the rate at which rapidly reversible blocking agents can be washed out of the oocyte recording chamber. In contrast, the cysteine-substituted AAAA channels (CAAA, ACAA, AACA, and AAAC) were all more strongly and persistently blocked by MTSEA. Average values of MTSEA block for the 0 position cysteine mutants were 63% for CAAA, 83% for ACAA, 90% for AACA, and 90% for AAAC (Fig 2 A). All of the testable -1 position cysteine substitutions were also persistently blocked by MTSEA, with average values of block being 32% for M392C/AAAA, 52% for F1144C/AAAA, and 67% for G1445C/AAAA. The G735C/AAAA either did not express or did not produce functional channels, since oocytes injected with this cRNA exhibited little or no inward Li+ current.
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The results of MTSEA block of WT EEEE channels and cysteine-substituted EEEE channels are summarized in Fig 2 B. WT EEEE channels were blocked by only 16 ± 3%, and this small block was reversed with wash. In contrast, all -1- and 0-position cysteine substitution mutants of the EEEE channel were persistently blocked by MTSEA. For these mutants, percent block values ranged from 83 to 92% for 0-position mutants, and from 62 to 80% for -1-position mutants. These results indicate that all eight -1- and 0-position side-chains project into the pore of the EEEE channel, as has been described previously (
Comparison of the MTSEA block patterns for EEEE and AAAA channels shows that all -1- and 0-position mutants were blocked by MTSEA in EEEE and AAAA channels, indicating that all of the tested positions are accessible to MTSEA in both channel types. Furthermore, within each motif, MTSEA block of the 0-position mutant was larger than that of the -1-position mutant for both EEEE and AAAA channels. These points of similarity in the MTSEA block patterns indicate that the orientations of amino acid side-chains at the 0 and -1 positions were not drastically altered by introduction of four alanines at the EEEE locus. In other details, the block patterns differed so that, for some cysteine substitution positions, fractional block by MTSEA differed in degree between the EEEE and AAAA channels. In comparing cysteine-substituted EEEE and AAAA channels at a given position, the differences in degree of fractional block by MTSEA are not surprising: with four small alanine side chains (-CH3) projecting into the pore in place of four larger, and charged, glutamate side chains (-CH2CH2COO-), substituted cysteine thiols may be somewhat differently accessible, or these thiols may be modified to a somewhat different degree, or thiol modification may not result in an identical degree of physical obstruction of the pore. The key finding, however, is that the EEEE and AAAA block patterns exhibited a degree of similarity that suggests that structural differences between the AAAA channel and WT are largely limited to the side-chain makeup of the EEEE locus.
High-affinity Block by External Ca2+ Occurs at the EEEE Locus
EEEE locus mutations have previously been shown to attenuate block by divalent cations entering the pore via its extracellularly oriented, or external, entrance (1C (
1 µM) was shifted
1050-fold by single alanine substitutions and
1,000-fold by substitution of alanine for all four EEEE locus glutamates (AAAA;
1C channels, step depolarizations in the presence of 2 µM FPL 64176 produced inward Li+ currents of long duration (Fig 3 A, top). Including 3 µM Ca2+ in the 100-mM Li+ solution bathing the extracellular surface of the patch (pipet solution) introduced frequent interruptions in the unitary inward currents, which represent individual Ca2+ block/unblock transitions. By contrast, inward Li+ currents through AAAA mutant channels were not detectably affected by 3 µM, or even 100 µM, external Ca2+ (Fig 3 A, bottom). When 1 mM Ca2+ was included in the patch pipet solution, a small degree of flicker block was observed. This presumably is the manifestation of Ca2+ binding to a low affinity site in the pore. These results coincide with those obtained at the whole-cell level (
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Examples of the high fluxes of both divalent (Ba2+) and monovalent (Li+) cations through WT 1C channels are illustrated in Fig 3 B (left). Fig 3 B (right) shows that, although Li+ is highly permeant in the AAAA channel, Ba2+ is not. In fact, lack of divalent cation permeability is characteristic of quadruple EEEE locus mutants, so that little or no inward current was observed with external Ba2+ or Ca2+ in the AAAA or QQQQ mutants (
Outward Ca2+ Channel Currents in Excised Patches
If there is a second high-affinity binding site for divalent cations located further from the external pore entrance than is the EEEE locus, then this deeper site could reasonably be expected to be accessible from the intracellular side of WT, AAAA, and QQQQ channels. We addressed this possibility by studying, in Ca2+ channel-bearing inside-out patches, block of outward Li+ currents by Ca2+ entering the pore from the internal (cytosolically oriented) pore entrance. For this set of experiments, we had greater success studying Ca2+ channels expressed in HEK293 cells than in oocytes. In inside-out patches from oocytes, with 100 mM Li+ in the bath (internal solution), outward single-channel Li+ currents were not readily observed. L-type Ca2+ channels are known to exhibit rapid rundown or loss of activity after patch excision, perhaps due to the loss of positive regulation by intracellular components (1215% of excised patches. Rundown, as well as residual block by patch-associated polyamines and Mg2+, was likely responsible for this low probability of observing unitary outward currents.
Five lines of evidence indicate that the single-channel currents that we recorded from inside-out patches were produced by transfected Ca2+ channels and not by ion channels endogenous to HEK293 cells. This is a particularly significant issue for the AAAA and QQQQ mutant channels, since their unitary conductance and block properties have not been previously described. First, consider the endogenous channels of HEK293 cells. Although Ca2+ channels native to HEK293 cells display some of the characteristics of L-type Ca2+ channels, these native channels are insensitive to the dihydropyridine agonist BayK 8644, and they were therefore not responsible for the long-duration openings that we studied (
Second, reversal potentials determined from whole-cell currentvoltage relationships were consistent with the overwhelming majority of current in transfected cells being attributable to exogenous Ca2+ channels (Fig 4 A). Average reversal potential for 1C-transfected cells was +31.2 ± 2.2 mV (n = 5), reflecting a significant preference in the WT
1C channel for Li+ over Cs+. AAAA-transfected cells had a reduced preference for Li+ over Cs+ (average reversal potential = +10.7 ± 1.3 mV, n = 6), which is consistent with the loss of selectivity caused by the quadruple EEEE locus mutations.
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Third, measurement of whole-cell currents in HEK293 cells demonstrated that the Li+ currents through single channels in excised patches were dominantly due to permeation of heterologously expressed Ca2+ channels, rather than endogenous channels. Fig 4 B shows that inward Li+ current density was 50-fold higher for cells expressing WT
1C compared with control cells (GFP-transfected only) and
8-fold higher for cells expressing AAAA compared with control. Likewise, outward Cs+ current densities were significantly greater in Ca2+ channel-transfected than in control cells (
17-fold for WT and
7-fold for AAAA; Fig 4 B).
Fourth, a dihydropyridine antagonist of L-type Ca2+ channels, nimodipine (10 µM), blocked inward Li+ currents through AAAA-transfected cells by 67 ± 8% (n = 3; -20 mV), which is similar to the degree of block of WT 1C channels expressed in oocytes (
1C-transfected cells, when micromolar Ca2+ was present in the bath, WT
1C channels were always blocked by Ca2+; there was no contamination with nonblocked channels. In the case of the AAAA-transfected cells, the absence of internal Ca2+ block of outward Li+ currents eliminates the possibility that currents recorded from these cells might have been carried by endogenous Ca2+ channels.
Internal Ca2+ Blocks Wild-Type Ca2+ Channels with High Affinity
Recordings of outward Li+ currents through single 1C channels in inside-out patches from HEK293 cells are shown in Fig 5 A. These channels exhibited multiple conductance states, a well-documented behavior of voltage-gated Ca2+ channels (e.g.,
1C channels were blocked when at least micromolar Ca2+ was present in the solution bathing the cytosolic surface of the patch. Representative WT single-channel records illustrate the flicker block that was introduced by internal Ca2+, with the number of flicker block events increasing with [Ca2+] (Fig 5 A). Quantitation of open and shut times yielded Ca2+ on rates (1/
open) that increased with [Ca2+] and off rates (1/
shut) that were concentration independent, findings that are indicative of a bimolecular reaction. At a membrane potential of +20 mV, the average off rate was 2,965 s-1 and the on-rate coefficient, determined as the slope of a linear regression fit, was 2.3 x 108 M-1 s-1. The WT channel exhibited high-affinity binding of Ca2+ in this configuration, as the apparent Kd is calculated to be
13 µM (koff/kon). The concentration-dependent on rate is similar to that of
1C channels.
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AAAA and QQQQ Channels Are Not Effectively Blocked by Internal Ca2+
In contrast to the case for WT channels, unitary outward Li+ currents through AAAA channels did not exhibit detectable flicker block with internal (bath) [Ca2+] as high as 1 mM (Fig 6), nor even 3 mM (data not shown). If Ca2+ block and unblock of AAAA channels occurred on a time scale shorter than the dead time of our recording system, then discrete Ca2+ block events would not be fully resolved and Ca2+ block would reduce unitary current amplitude in a manner graded with [Ca2+]. A potentially complicating factor is that, like WT, the AAAA mutant exhibited four clear conductance levels. However, no graded reductions in AAAA channel unitary current amplitudes were detected as internal Ca2+ was increased, so that AAAA channels exhibited the same unitary current amplitudes in the presence of 1 mM internal Ca2+ as in control internal solution. Thus, measurements of unitary current amplitudes also did not reveal block by internal Ca2+ of outward Li+ currents in AAAA channels.
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QQQQ is another EEEE locus quadruple mutant that lacks high-affinity binding of Ca2+ when probed via the pore's external entrance (
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DISCUSSION |
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Side-Chain Orientation in the AAAA Mutant
Because high-resolution structures are not available for the pores of mutant or WT Ca2+ channels, the effects of mutations upon pore structure can only be inferred from indirect information such as altered pore block. This is one reason why the idea that Ca2+ channels possess only a single locus of high-affinity binding in their pore-lining regions has remained tantalizingly short of conclusive.
We have tried to determine whether amino acid substitutions in the EEEE locus have little effect on structure other than to replace one side chain with another while preserving general spatial organization, or whether substitutions may have in addition the undesired effect of significantly altering side-chain orientation: in one extreme example, side-chains that project into the lumen of the WT pore to interact with permeating ions might, upon amino acid substitution, project away from the lumen of the mutant pore. Even more problematic, it is possible that EEEE locus substitutions could conceivably cause significant reorientation of side chains outside the EEEE locus. To address this kind of problem, we investigated the accessibility of substituted cysteines to determine side-chain orientation in the pore, but one view of this approach is that relying on additional mutagenesis work in the absence of "real" structural information only compounds the problems inherent in the earlier mutagenesis work. This may be true. Yet atomic-scale pore structures for both WT and mutant Ca2+ channels are not likely to be in hand any time soon, and further mutagenesis work allows comparison of the effects of various mutations, thereby providing tests for the consistency of conclusions regarding pore structure. Following this logic, we have examined whether side chains that have been found in previous mutagenesis work to project into the pore of WT channels also do so in the relatively severe AAAA mutant.
Based on substituted-cysteine accessibility analysis of WT channels (Wu, X., H.D. Edwards, and W.A. Sather, manuscript submitted for publication) and on evidence that the EEEE locus side-chains together form a single protonatable site in the pore (
Because we were specifically concerned about the possible existence of Ca2+ binding sites located internally to the EEEE locus, we did not examine side-chain orientation at more external positions (+1, +2) in the AAAA channel. Examination of substituted-cysteine accessibility at deeper pore positions was not attempted, based on suspected large-scale structural similarity with the KcsA pore structure (
Considering all of the above, the most probable conclusion that can be drawn from the substituted-cysteine accessibility work is this: despite the limited nature of the information provided, the concern that fourfold alanine substitution at the EEEE locus might result in substantial rearrangement of pore structure seems unlikely owing to the absence of a drastic affect on structure near and at the EEEE locus. Even so, a viable alternative view is that the small structural differences detected between EEEE and AAAA channels hint at larger structural differences deeper in the pore. Identification of sequences lining the deeper pore will allow the more extensive comparison of pore structure between EEEE and AAAA channels needed to address this lingering concern.
In contrast to the situation with cysteine-substituted mutants, the modest block by MTSEA of the noncysteine-substituted AAAA channel was reversible, and occurred at a rate similar to that of Cd2+ unblock of Ca2+ channels in oocytes. One reasonable interpretation of these observations is that the cationic MTSEA is attracted into the pore, where it obstructs permeant ion flow. Whatever the mechanism of this kind of block by MTSEA, however, block did not result from covalent attachment of -SCH2CH2NH3+ to the noncysteine-substituted AAAA channel, and so this kind of block was separable from that measured for the cysteine-substituted mutants.
The Number of High-Affinity Binding Sites Involved in Selective Ion Permeability
Our examination of AAAA and QQQQ channels for block of outward Li+ current by Ca2+ entering the pore via its intracellularly oriented entrance showed that Ca2+ at up to 3 mM failed to effectively block these mutants. Specifically, we were unable to detect the kind of discrete Ca2+ block and unblock events that are the kinetic signature of high-affinity Ca2+ binding. Thus, Ca2+ channels lacking an intact EEEE locus are unable to bind Ca2+ with approximately micromolar affinity, whether Ca2+ enters the pore via the external entrance, as shown in previous work, or via the internal entrance, as shown here. Combining these results with substituted-cysteine accessibility results suggesting that pore structure was not extensively disrupted in the AAAA mutant solidifies the view that the EEEE locus comprises the only high-affinity Ca2+ binding site in the pore of voltage-gated Ca2+ channels. Our results are consonant with those obtained in studies of double alanine substitution mutants, which are permeated by at least one divalent cation, Ba2+, but that exhibit greatly weakened Ca2+ block and binding (
In the AAAA and QQQQ mutants, 1 mM internal Ca2+ not only failed to produce resolvable, discrete block/unblock events, but this very high level of Ca2+ also failed to produce a detectable change in unitary Li+ current amplitude. Two-electrode voltage-clamp measurements in oocytes have shown that currents carried by 100 mM Li+ through AAAA and QQQQ channels are half-blocked by 1.2 mM Ca2+ (
50% at 1 mM Ca2+, despite the absence of any high-affinity Ca2+ binding site. However, the recording conditions and solutions differ between two-electrode voltage-clamp and inside-out patch work, shifting the half-block value for WT from the 1-µM value measured by two-electrode voltage clamp to 13 µM, measured here with inside-out patches. Scaling the mutant channel half-block values by this factor of 13 predicts that mutant channels would only be blocked by 6% at 1 mM Ca2+, a reduction in unitary current amplitude so small that we were unable to detect it.
An External Low-Affinity Ca2+ Binding Site?
We were unable to detect discrete block events in AAAA channels when Ca2+ entered the pore from the internal entrance, but we did detect what appeared to be discrete block events when Ca2+ entered the AAAA pore via the external entrance. Although it was difficult to separate the external block events from channel gating events, there appeared to be more resolved open/shut transitions in high Ca2+ than in low Ca2+ (Fig 3 A). Assuming that the brief open/shut transitions produced in high external Ca2+ represent block of Li+ flux by Ca2+ binding in the permeation pathway, where could such binding occur in AAAA channels that lack an EEEE locus? A speculative possibility is that Ca2+ binds to a low-affinity site just external to the EEEE locus. Such a site would necessarily possess low Ca2+ binding affinity because the AAAA channel is only weakly blocked by Ca2+; an external localization is plausible based on the fact that Ca2+, which is impermeant in the AAAA channel, produced discrete block only when present on the external side of the membrane. Detection of discrete block events is possible for a low-affinity site assuming that a large majority of block events are too brief to be resolved, and that only the rare, very longest events in the distribution of blocked-state lifetimes are detected.
The low-affinity block by Ca2+ of monovalent cation flux through AAAA channels (IC50 1 mM) has been thought, since it was first observed, to be mediated by a superficial, externally localized site (
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Footnotes |
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1 Abbreviations used in this paper: GFP, green fluorescent protein; MTSEA, 2-aminoethyl methanethiosulfonate; WT, wild type.
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Acknowledgements |
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We thank Tsutomu Tanabe, Veit Flockerzi, and Franz Hofmann for gifts of Ca2+ channel cDNAs, and Emily Liman for the gift of the pGEMHE vector.
This work was supported by grant NS35245 (W.A. Sather) and fellowship MH11717 (S.M. Cibulsky) from the National Institutes of Health.
Submitted: 28 March 2000
Revised: 6 July 2000
Accepted: 7 July 2000
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