ACCELERATED PUBLICATION
Molecular Determinants of Ion Permeation and Selectivity in Inositol 1,4,5-Trisphosphate Receptor Ca2+ Channels*

Darren BoehningDagger §, Don-On Daniel Mak, J. Kevin Foskett, and Suresh K. JosephDagger ||

From the Dagger  Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19103 and the  Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, February 20, 2001




    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca2+ release channel (inositol 1,4,5-trisphosphate receptors (IP3Rs)) that is gated by the second messenger inositol 1,4,5-trisphophate (IP3). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a 45Ca2+ flux assay and single channel electrophysiological analyses. Two IP3R mutations, V2548I and D2550E, retained the ability to release 45Ca2+ in response to IP3. When analyzed at the single channel level; both recombinant channels had IP3-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K+ conductance (489 ± 13 picosiemens (pS) for V2548I versus 364 ± 5 pS for wild-type), but retained a Ca2+ selectivity similar to wild-type channels (PCa2+:PK+ ~ 4:1). Conversely, D2550E channels were nonselective for Ca2+ over K+ (PCa2+:PK+ ~ 0.6:1), while the K+ conductance was effectively unchanged (391 ± 4 pS). These results suggest that amino acid residues Val2548 and Asp2550 contribute to the ion conduction pathway. We propose that the pore of IP3R channels has two distinct sites that control monovalent cation permeation (Val2548) and Ca2+ selectivity (Asp2550).




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

In response to a wide variety of external stimuli, the second messenger inositol 1,4,5-trisphosphate (IP3)1 is generated from the phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate (1). IP3 then diffuses through the cytosol and binds to the IP3 receptor (IP3R), an intracellular ion channel that mediates the release of Ca2+ from the endoplasmic reticulum. IP3R-mediated increases in cytoplasmic [Ca2+] modulate a diverse array of cellular processes (2).

The IP3R channel is composed of four ~300-kilodalton subunits that together form a single ion conduction pore (3). The C terminus of each subunit is believed to contain six transmembrane helices and is separated from the N-terminal IP3 binding domain by a large intervening cytoplasmic region (4). The basic six transmembrane domain topology of IP3Rs is shared with other cation channels, including the superfamily of voltage-gated ion channels, cyclic nucleotide-gated (CNG) ion channels, vanilloid receptors, and other members of the trp family of ion channels. By analogy, it has been suggested that putative transmembrane helices 5 and 6 and the intervening intraluminal loop constitute the ion permeation pathway of IP3Rs (5, 6), a proposal that has been experimentally verified by deletion mutant analysis (7). The intraluminal loop of both IP3Rs and ryanodine receptors (RyRs) contain a stretch of amino acids, GVGD and GIGD, respectively, that may be homologous to the selectivity filter of K+ channels. However, there is no direct evidence that residues within this region contribute to the ion conduction pathway of IP3Rs. The present study is the first to identify key residues that regulate ion permeation and selectivity in IP3R channels. We mutated three amino acids in this region and examined the functional consequences using a 45Ca2+ flux assay and single channel electrophysiological analyses, carried out by patch clamping nuclei isolated from transfected COS cells.2 Two mutants (V2548I and D2550E) of the rat type 1 IP3R retained the ability to release 45Ca2+ in response to IP3. When analyzed at the single-channel level, mutant V2548I channels had a larger K+ conductance compared with wild-type channels with no alteration in the selectivity for divalent cations. However, substitution of Asp2550 with Glu resulted in channels that were nonselective for divalent cations without affecting K+ conductance. We propose that the molecular mechanism for divalent cation selectivity in IP3Rs involves a ring of four aspartic acid residues at position 2550 that form a Ca2+ binding site within the ion conduction pathway, with ion conductance determined by valine at position 2548. Divalent cation interaction with two sites may enable multi-ion occupancy and high ion throughput in the IP3R.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Expression Constructs-- The neuronal rat type I SI(-), SIII(+), SII(+) splice variant in pCMV was the kind gift of Dr. Thomas Südhof (University of Texas Southwestern Medical Center). This cDNA was cloned into pcDNA3.1 as described elsewhere (9). Mutations of Asp2550 (rat) to Glu, Asn, and Ala have been described previously (9). All other mutations were carried out using the QuickChange site-directed mutagenesis kit as per manufacturer's instructions (Stratagene, La Jolla, CA). Sequences of the mutagenic primers used for the polymerase chain reactions are available upon request.

Cell Culture and Transfection-- COS-7 cells were maintained and transfected as described previously (9).

45Ca2+ Flux Measurements-- 45Ca2+ flux assays were performed exactly as described previously (9, 10). Briefly, microsomal vesicles were prepared from COS-7 cells transiently transfected with either wild-type or mutated IP3R constructs in conjunction with SERCA-2b (human). The vesicles were then assayed for 45Ca2+ uptake in the presence of potassium oxalate and MgATP. The inclusion of IP3 in the assay buffer caused a reduction in the initial rate of SERCA-dependent uptake, giving an indirect measure of IP3R activity. This assay has been shown to measure 45Ca2+ flux only through recombinant IP3Rs (9, 10). The initial rate of uptake in the presence of IP3 is presented as a percentage of the rate measured without IP3.

Preparation of Cellular Homogenates for Patch Clamp Recording-- Preparations of cellular homogenates for patch clamp recording of nuclei are described in detail elsewhere.2 Briefly, cells were gently homogenized in a motor-driven glass Teflon homogenizer in a buffer containing 0.25 M sucrose, 0.15 M KCl, 3 mM beta -mercaptoethanol, and 10 mM Tris, pH 7.5. Cell integrity was monitored by trypan blue exclusion, and homogenization was allowed to proceed until ~30% of cells were lysed. Cell lysates were stored in the same buffer on ice and used on the same day in patch clamping experiments.

Patch Clamping COS-7 Cell Nuclei-- Approximately 10 µl of cellular homogenate was added to a dish containing 1 ml of bath solution (see "Patch Clamp Solutions") and transferred to the stage of a microscope for patch clamping. Isolated nuclei, visually free of extraneous cellular debris, were localized by trypan blue staining and patch-clamped at room temperature. Single channel currents were amplified using an Axopatch-1D amplifier (Axon Instruments, Foster City, CA) with anti-aliasing filtering at 1 kHz and transferred to a Power Macintosh 8100 via an ITC-16 interface (Instrutech Corp., Port Washington, NY). Data were digitized at 5 kHz and written directly to hard disc by Pulse + PulseFit software (HEKA Elektronik, Lambrecht/Pfalz, Germany). Single channel recordings were analyzed using TAC 3.03 (Bruxton, Seattle, WA) and plotted using Igor Pro 3 (WaveMetrics, Lake Oswego, OR) and SigmaPlot (SPSS Science, Chicago, IL). Permeability ratios were calculated using the experimentally determined reversal potentials as described elsewhere (11).

Patch Clamp Solutions-- Bath solution contained 140 mM KCl, 10 mM HEPES, 500 µM BAPTA, 0.001% trypan blue, and 250 nM [Ca2+]free adjusted to pH 7.1 with KOH. An additional 5 mM K+ ions was contributed to the solution by the adjustment of the pH. Pipette solutions contained 140 mM KCl, 10 mM HEPES, 100 µM BAPTA, 0.5 mM NaATP, 10 µM IP3, and 1.0 µM [Ca2+]free adjusted to pH 7.1 with KOH. Free calcium concentrations in all buffers were determined using a Ca2+-selective mini-electrode as described previously (12). For determinations of ionic selectivity, high Ca2+ bath buffer contained 50 mM CaCl2, 30 mM KCl, 10 mM HEPES adjusted to pH 7.1 with KOH. Low K+ pipette solutions contained 14 mM KCl, 10 mM HEPES, 100 µM BAPTA, 0.5 mM NaATP, 10 µM IP3, and 1.0 µM [Ca2+]free adjusted to pH 7.1 with KOH. All selectivity determinations were corrected for the liquid junction potential as described previously (13).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The predicted transmembrane topology of the IP3R and the putative location of the three residues, Val2548, Asp2550, and Asp2569, examined in the present study are diagrammed in Fig. 1A. All three amino acids are within a predicted intraluminal loop between transmembrane helices 5 and 6, a region that contains the pore domain in other cation channels. Within this region is a sequence GVGD, which is highly reminiscent of the GYGD signature sequence of the selectivity filter of K+ channels. The Tyr residue in K+ channels plays a critical role in defining the size of the pore and thereby providing K+ selectivity (14). In contrast, the selectivity filters of Ca2+-selective ion channels lack the two central residues (YG) present in the K+-selective channels (Fig. 1A). Nevertheless, the permeation properties of the IP3R are more similar to those of Ca2+ channels, including selectivity for divalent cations, lack of selectivity for monovalent alkali metal cations, and block by divalent cations including Mg2+ (15). The similar permeation properties of IP3R, CNG, and voltage-gated Ca2+ channels suggest that the IP3R channel pore likely contains a high affinity binding site for Ca2+ ions. In CNG- and voltage-gated Ca2+ channels, this site is conferred by an acidic residue equivalent in sequence to Asp2550 in the IP3R channel (Fig. 1A). Because the IP3R permeation properties are reminiscent of those of other Ca2+ channels, but the putative pore sequence is more closely related to K+ channels, we examined the effects of mutations of Val2548 and Asp2550 on the permeation properties of the IP3R channel. Based on the roles of these residues in K+ and Ca2+ channels, respectively, we hypothesized that Val2548 might influence conduction by its participation in the molecular interactions that control the size of the pore, whereas Asp2550 may determine divalent cation selectivity. In addition, we also mutated Asp2569, because mutation of the homologous residue in the RyR influenced its permeation properties (16).



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Fig. 1.   Mutations in the IP3R C terminus affect 45Ca2+ flux. A, each monomer of an IP3R channel contains six transmembrane helices, with the pore of the channel believed to be formed by contributions from residues located between transmembrane helices 5 and 6 (7, 9). A hydrophobic segment immediately preceding the selectivity filter may be a pore helix, by analogy with the KcsA K+ channel (14). Residues Val2548, Asp2550, and Asp2569 were subjected to site-directed mutagenesis and are indicated in bold on the schematic and by arrows and residue number on the expanded sequence. The N terminus, which comprises the majority of the IP3R protein, has been omitted from the schematic. The sequence alignment of the pore region of the type I inositol trisphosphate receptor (IP3R-1), rabbit skeletal muscle ryanodine receptor (RyR-1), Shaker potassium channel, vanilloid receptor (VR1), bovine rod cyclic nucleotide-gated channel (bROD), repeat IV of the T-type Ca2+ channel (T-type IV), and repeat IV of the L-type Ca2+ channel (L-type IV) is shown. Darker shading indicates identity between all seven ion channels, and the gray shaded residues indicate homology. The putative selectivity filter of all channel families is indicated by a solid line. GenBankTM accession numbers (from top to bottom) are J05510, X15750, XM_008512, M17211, X51604, NM_018896, and L04569. B, COS cells were transfected with wild-type (WT) and mutant IP3R constructs, and 20 µg of total cellular lysate was loaded onto a 5% SDS-polyacrylamide gel electrophoresis gel. 20 µg of cerebellum microsomes were loaded as a positive control. IP3R bands were detected by immunoblotting with an antibody specific to the C terminus of the type I isoform (8). There was no significant difference in expression levels over multiple experiments (data not shown). C, microsomes were prepared from COS cells expressing recombinant IP3Rs and SERCA-2b. To measure Ca2+ flux through the recombinant channels, the initial rate of ATP-dependent 45Ca2+ uptake into the COS cell microsomes was measured in the presence of 1.0 µM IP3. Uptake rates were plotted as a function of control rate of uptake in the absence of IP3. Therefore, Ca2+ release through the recombinant channels would be indicated by a reduction in the initial rate of uptake when compared with control. Absence of response to IP3 is indicated by a solid line. The 45Ca2+ release activities of mutations D2550E, D2550N, and D2550A have been reported previously (9). *, significantly different from control (p < 0.001).

Val2548 was mutated to Ile or Tyr, the amino acids present at the analogous position in ryanodine receptors and K+ channels, respectively (Fig. 1A). Asp2550 and Asp2569 were mutated to Glu, Asn, or Ala. All the mutant IP3R channels were expressed at levels comparable with the wild-type channel when transiently transfected into COS-7 cells (Fig. 1B). Mutant channels were first screened for Ca2+ release activity by measurements of IP3-dependent 45Ca2+ flux from microsomes prepared from COS cells co-transfected with both SERCA2b and IP3R cDNAs (9). Mutating Val2548 to Ile did not alter channel function in this assay, whereas no Ca2+ release activity was observed with substitution by Tyr (Fig. 1C). As shown previously, Glu (but not Asn or Ala) substituted for Asp2550 in the Ca2+-release assay (9). Asp2569, predicted to be the final residue before the beginning of the putative transmembrane helix 6 (Fig. 1A), was intolerant to substitutions (Fig. 1C). Mutation of the analogous position (D4917A) in the RyR type 1 channel also abolished Ca2+ permeability (16).

Patch clamp electrophysiology of transfected COS cell nuclei was performed to examine the effects of the mutations on ion permeation. Our protocol enables recording of recombinant IP3R channels specifically without contributions from endogenous IP3R channels.2 K+ was used as the permeant ion to minimize Ca2+-dependent inactivation and maximize single channel conductance (17). Using these recording conditions, IP3-activated channels were only detected in nuclear membrane patches from cells expressing wild-type, V2548I, or D2550E channels (data not shown). Open probabilities determined at 1 µM free Ca2+ were similar for both mutant channels and wild-type channels (wild-type, 0.59 ± 0.07; V2548I, 0.65 ± 0.10; D2550E, 0.68 ± 0.13).

From analyses of current-voltage relationships determined in symmetrical 140 mM KCl, the slope conductance of V2548I channels was 489 ± 13 pS, which was significantly larger than that of wild-type channels (364 ± 5 pS). In contrast, the ion selectivity of the V2548I channel was unchanged compared with that of the wild-type channel. The monovalent cation:anion selectivity, determined from reversal potentials measured in the presence of a 10-fold KCl gradient (14 mM KCl pipette, 140 mM KCl bath) (11), was identical for V2548I and wild-type channels (Erev = +44.9 ± 0.5 mV; PK/PCl = 22:1; Fig. 1C). To determine the Ca2+ selectivity, channels were first detected in symmetrical 140 mM KCl, and then the bath was replaced with a high Ca2+ buffer (50 mM CaCl2, 30 mM KCl). Under these conditions, V2548I channels had a Erev of +19.3 ± 1.8 mV, corresponding to PCa/PK ~ 4.1 (Fig. 3D), not significantly different from that observed for wild-type IP3R channels (data not shown)2. Thus, these results demonstrate that mutation of Val2548 to Ile increased channel conductance but was without effect on the ion selectivity properties of the pore.

The conductance of D2550E channels was similar to that of wild-type channels (391 ± 4 pS versus 364 ± 5 pS, respectively; Fig. 2B). The mutation was also without effect on the cation:anion selectivity (PK/PCl = 22:1; Fig. 1C). In contrast, the mutant channel had a significantly altered cation selectivity. Erev for the D2550E channel in the presence of a Ca2+ gradient was shifted to -7.4 ± 2.8 mV (Fig. 2D), giving a calculated PCa/PK of 0.63. Thus, D2550E channels have similar conductance properties as wild-type channels, but they have largely lost the ability to discriminate between K+ and Ca2+.



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Fig. 2.   Mutations within the putative pore region affect conductance and Ca2+ selectivity. A, representative single-channel recordings of wild-type, V2548I, and D2550E IP3R channels measured by patch clamp of isolated COS cell nuclei in the presence of symmetrical 140 mM KCl (see "Experimental Procedures").2 Zero current level is indicated by an arrow, and the holding potential is listed above each trace. B, current-voltage plot of the wild-type (closed circles), D2550E (open triangles), and V2548I (open circles) IP3R channels in symmetrical 140 mM KCl. Solid lines represent the linear regression through the data points. The slope conductance of the wild-type, D2550E, and V2548I channels was 364 ± 5, 391 ± 4, and 489 ± 13 pS, respectively. C, the current voltage relationship was measured in the presence of a 10-fold KCl gradient (14 mM KCl pipette, 140 mM KCl bath; see "Patch Clamp Solutions") to determine cation:anion selectivities. The wild-type (closed circles), D2550E (open triangles), and V2548I (open circles) channels had identical reversal potentials (+44.9 ± 0.5 mV) under these ionic condition, corresponding to a permeability ratio for K+ over Cl- of 22:1. Identical results were obtained when a voltage ramp was utilized to determine Erev (as in D; data not shown). Solid lines represent linear regressions through data points. D, the Ca2+ selectivity of V2548I and D2550E channels was determined by exchanging the bath with a high Ca2+ buffer (50 mM CaCl2, 30 mM KCl) after first detecting channels in symmetrical 140 mM KCl. A voltage ramp from -80 mV to +80 mV over 1 s was then employed to determine Erev. Zero current level is indicated by a solid line, and opening events were fit with a dashed line. The values of Erev and the permeability ratios for the representative experiment are given. The average Erev of at least three separate determinations as well as the corresponding permeability ratios are given in the text.

The present study has identified two residues (Val2548 and Asp2550) that, when mutated, affect the conductance or divalent cation selectivity of the IP3R channel. Thus these two residues are likely to be part of the ion conduction pathway of IP3Rs. This result, taken together with analyses of IP3R topology (19, 20) and the sequence homology with the selectivity filter of K+ channels, confirms previous predictions that the pore region of the IP3R is constructed in a manner analogous to that of several classes of voltage- and ligand-gated cation channels with the pore located between the last two transmembrane segments (5, 20). A similar conclusion was reached for the RyR channel (21).

In CNG- and voltage-gated Ca2+ channels, a conserved glutamate in the pore region is critical for Ca2+ selectivity (22, 23). Cysteine accessibility and pH sensitivity studies of these channels have suggested that the side chains of four of these residues (contributed by each repeat or monomer) likely project into the pore to coordinate the permeant Ca2+ ion and regulate divalent cation permeation (24, 25). The location of Asp2550 in the analogous position in the IP3R channel and the effects of its mutation on divalent cation selectivity indicate that this residue functions similarly in the IP3R channel. The fact that mutation of this residue to Glu with preservation of its charge affected the Ca2+ selectivity suggests that steric considerations as well as electrostatic forces contribute to the positioning of the ring of carboxylate groups in the pore. Functional non-equivalence of Glu and Asp residues at analogous positions in voltage-gated Ca2+ channels and CNG channels has also been noted previously (23, 26). One of the findings of the present study is that although the D2550E mutant exhibited diminished divalent cation selectivity, permeation of monovalent cations was unaffected, suggesting that a high affinity interaction at position 2550 is not rate-limiting for monovalent ion permeation. Similarly, monovalent ion permeability is not impaired when all four glutamates within the EEEE locus are mutated to Ala or Gln in voltage-gated Ca2+ channels (27). This is also the observed result when the analogous Asp is mutated to Asn in vanilloid receptors (28).

The conserved acidic residue that is critical for divalent cation selectivity and permeation in Ca2+-selective channels is adjacent to the GXG signature sequence in other cation channels (Fig. 1A). The crystal structure of the Streptomyces lividans K+ channel indicates that this acidic residue is exposed at the outer mouth of the pore (14). A distinction between the IP3R channel on one hand, and CNG channels and voltage-gated Ca2+ channels on the other, is that the latter have deletions of two amino acids (Fig. 1A) between the initial glycine and the acidic residue of the signature sequence, whereas the IP3R channel contains them. The crystal structure of the K+ channel has shown that the rigid arrangement of backbone carbonyl oxygen atoms of the selectivity filter within the GYG sequence is responsible for the precise coordination of K+ ion in the pore (14). In this structure the side chain of the central tyrosine residue is directed away from the pore and makes specific interactions with other parts of the protein that contribute to holding the pore at a fixed size (14). Mutation of the GVG sequence in IP3R to the GYG sequence of K+ channels resulted in an absence of detectable IP3R channel activity. On the other hand, mutation to GIG increased the K+ conductance. It is interesting to note that the single channel conductance of RyR, which contains the GIG sequence, is greater than that of IP3R (16, 18) and that the reverse mutation of Ile in the RyR to Val, as in IP3R, confers a lower conductance on RyR channels (16). Thus, this residue likely plays a role in IP3R channels in contributing to the mechanisms that determine the size of the pore, which in turn contribute to the magnitude of alkali metal cation conductance.

We propose a model in which the pore of IP3R channels has two distinct sites that control K+ ion permeation (Val2548) and divalent cation selectivity (Asp2550). The interaction of permeant ions with two distinct sites in the IP3R pore may enable multi-ion occupancy of the pore, which could promote high throughput permeation rates by electrostatic destabilization.


    ACKNOWLEDGEMENT

We thank Z. Lu for comments on the manuscript.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01-DK34804 (to S. K. J.) and R01-MH59937 (to J. K. F.), a predoctoral fellowship from training Grant T32-AA07463 (to D. B.) from the National Institutes of Health, and by American Heart Association Grant 9906220U (D.-O. D. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Neuroscience, Johns Hopkins University, 725 N. Wolfe St., Rm. 813 WBSB, Baltimore, MD 21205.

|| To whom correspondence should be addressed: Dept. of Pathology and Cell Biology, Thomas Jefferson University, 1020 Locust St., Rm. 230A, JAH, Philadelphia, PA 19107. Tel.: 215-502-1222; Fax: 215-923-6813; E-mail: suresh.joseph@mail.tju.edu.

Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.C100094200

2 D. Boehning, S. K. Joseph, D. D. Mak, and J. K. Foskett, submitted for publication.


    ABBREVIATIONS

The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; RyR, ryanodine receptor; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CNG, cyclic nucleotide-gated; S, siemens.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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