Correspondence to: Michael Fill, Loyola University Chicago, Department of Physiology, 2160 S. First Avenue, Maywood, IL 60153-5500., mfill{at}luc.edu (E-mail), Fax: 708-216-5158; (fax)
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The inositol 1,4,5-trisphosphate receptor (InsP3R) forms ligand-regulated intracellular Ca2+ release channels in the endoplasmic reticulum of all mammalian cells. The InsP3R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP3R pore. Mutant InsP3Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; PCa/PCs = 6.3). These mutant channels bound InsP3, but ligand occupancy did not regulate the constitutively open pore (Po > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 23982589) near the COOH terminus of the protein forms the InsP3R pore. Further, we have produced a constitutively open InsP3R pore mutant that is ideal for future site-directed mutagenesis studies of the structurefunction relationships that define Ca2+ permeation through the InsP3R channel.
Key Words: intracellular Ca2+ release, Ca2+ signaling, recombinant channel
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The inositol 1,4,5-trisphosphate receptor (InsP3R)1 gene family encodes a highly homologous group of proteins localized to the endoplasmic reticulum (ER). The InsP3R gene family has three members (types 1, 2, and 3) that are ubiquitously expressed in metazoans (
A first step towards understanding structurefunction relationships in any protein is to locate the primary sequences that associated with its general features, such as ligand binding and/or transmembrane regions (TMRs). Analysis of the recombinant InsP3R revealed that this protein is composed of three domains: the NH2-terminal InsP3-binding domain, the COOH-terminal channel domain, and the central coupling domain (
The InsP3R protein is thought to tetramerize to form functional Ca2+ release channels (
It is reasonable to hypothesize that the fifth and sixth TMR and the interceding loop are the most likely region of the InsP3R to form the trans-ER ion permeation pathway. To experimentally test this hypothesis, single channel function of different type 1 InsP3R TMR-deletion mutants was defined in planar bilayer studies. Our data indicate that the fifth and sixth TMRs and the interceding loop contain important structural determinants of the InsP3R channel's permeation pathway that govern its conduction and selectivity. Our data also suggest that the 14 TMR region of the pInsP3R contains important sequence and/or structural elements that regulate gating of the pore.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
[3H]InsP3 (21 Ci/mmol) was obtained from Du Pont-New England Nuclear. Unlabeled InsP3 was purchased from LC Laboratories Inc., and heparin was from Sigma Chemical Co. Ryanodine was purchased from Calbiochem Corp. Lipids, L--phosphatidylcholine, L-
-phosphatidylethanolamine, and L-
-phosphatidylserine were obtained from Avanti Polar Lipids.
Plasmid Construction and Expression
The full-length type 1 plasmid (pInsP3R-T1) was assembled from overlapping cDNA clones isolated from a rat brain library as previously described (1-4 encoded a protein missing residues 22112416. The expression plasmid pInsP3R
5-6 encoded a protein missing residues 23982589. Construction strategies of these two expression plasmids is described in detail elsewhere (Galvan, D., E. Borrego-Diaz, P.J. Perez, and G.A. Mignery, manuscript submitted for publication). Galvan and co-workers denoted the plasmids as TMR5-6+C and TMR1-4+C, respectively. Here, the plasmids (pInsP3R-T1, pInsP3R
1-4, and pInsP3R
5-6) were transiently transfected into COS-1 cells. COS-1 cells were transfected with each plasmid or sheared salmon sperm (SS) DNA using the DEAE-dextran method as described by
CHAPS Solubilization and Gradient Sedimentation
COS-1 cells transfected with either pInsP3R-T1, pInsP3R1-4 and pInsP3R
5-6, or the SS DNA were harvested 4872 h after transfection, and microsomes were prepared as described previously (
InsP3 Binding Assays
[3H]InsP3 ligand binding assays were performed as previously described (
Single Channel Assay
Planar lipid bilayers were formed across a 150-µm diameter aperture in the wall of a Delrin partition as described (
where R, T, and F have their usual meanings. Note that [Ca]trans/[Ca]cis was equal to one.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression and Isolation of Type 1 InsP3R TMR-deletion Mutants
The full-length (pInsP3R-T1) and TMR-deletion (pInsP3R1-4 and pInsP3R
5-6) mutants were transfected into COS-1 cells using the DEAE-dextran method (
1-4, and pInsP3R
5-6 plasmids were Western blotted with antibodies directed against the NH2 and COOH termini of the receptor. Blots performed with the COOH-terminus antibody are shown in Figure 1 C. These data indicate that the expressed InsP3R proteins were of the expected size and targeted to the correct microsomal fraction. It is possible that over expression of recombinant InsP3R may induce elevated expression of endogenous receptor. This possibility was thoroughly examined and dispelled in a previous study (
|
InsP3 Binding of the Type 1 InsP3R TMR-deletion Mutants
Equilibrium InsP3 binding assays were performed using microsomal proteins from transfected COS-1 cells (Table 1). The full-length recombinant receptor (pInsP3R-T1) and both TMR-deletion mutants (pInsP3R1-4 and pInsP3R
5-6) bind significant amounts of InsP3. The SS DNA control microsomes did not bind InsP3 at significant levels above nonspecific background. The amount of InsP3 bound was normalized to the relative protein expression of each InsP3R construct by densitometry. These results are consistent with previous studies in which microsomes of transfected COS-1 cells contained abundant amounts of immunoreactive receptor protein and bound significant amounts of [3H]InsP3 (
|
Single Channel Properties of the Type 1 InsP3R TMR-deletion Mutants
The TMR-deletion mutant InsP3R receptor proteins were incorporated into proteoliposomes for fusion into planar lipid bilayer studies. Microsomes from COS-1 cells transfected with either pInsP3R-T1, pInsP3R1-4, pInsP3R
5-6, or control SS DNA were solubilized in CHAPS detergent and sedimented over 520% sucrose density gradients. The tetrameric receptor complex (i.e., the channel complex) migrates to a position on the gradient beyond the majority of other proteins (
-phosphatidylcholine and L-
-phosphatidylserine containing liposomes as described previously (
No detectable InsP3/heparin-sensitive Cs+ conducting channels were incorporated into the bilayer after fusion of proteoliposomes containing gradient receptor fractions from nontransfected COS-1 cells, control (SS DNA)-transfected cells, or pInsP3R5-6transfected cells. Incorporation of proteoliposomes containing the pInsP3R
1-4 construct resulted in the appearance of a high conductance (~300 pS) ion channel with high open probability (>0.80). Sample single channel activity from the pInsP3R
1-4 channel is shown in Figure 2. The pInsP3R
1-4 channel was nearly always open with frequent and usually brief (~1 ms) flickers to the close state. Long closed events (>20 ms) were rare. Single channel activity was observed in the presence (Figure 2 A) and absence (Figure 2 B) of InsP3. Single channel activity was also not impacted by the addition of 10 µM ryanodine or 50 µg/ml heparin (Figure 2 C). Corresponding total amplitude histograms under each experimental condition are also presented in Figure 2. The channel was open most of the time with frequent but brief transitions to the closed state. Thus, these data suggest that the pore formed by the pInsP3R
1-4 protein was not modulated by agents (i.e., InsP3 and heparin) that modulate function of wild-type InsP3R channels. Additionally, the pInsP3R
1-4 pore was constitutively open (i.e., high Po, n = 6). Under optimal experimental conditions, the Po of full-length type 1 InsP3R channels is relatively low (
|
The permeation properties of the pInsP3R1-4 pore were also defined. Stationary single channel activity was recorded for extended periods (~5 min) at several different membrane potentials. The unitary current amplitude (Cs+ charge carrier) was measured as a function of membrane potential. Sample single channel records at different membrane potential (0, 20, and 40 mV) are shown in Figure 3 A. A sustained high Po was a fundamental feature the pInsP3R
1-4 pore at all membrane potentials tested. The activity of this mutant channel was voltage dependent. For example, the Po increased from ~85 to 95% when the membrane potential was changed from 0 to 40 mV. This modest voltage dependency of channel activity appears to be a persistent and consistent feature unique to the mutant InsP3R channel.
|
The unitary Cs+ current carried by the pInsP3R1-4 pore was Ohmic with a slope conductance of 284 pS (n = 9). Unitary Cs+ current reversed at -22 mV, indicating that the pInsP3R
1-4 pore was cation selective (Figure 3 B). The unitary Ca2+ current was also Ohmic at relatively large negative membrane potentials, with a slope conductance of 60 pS (Figure 3 C; n = 7). The selectivity of the channel was probed under biionic conditions (Figure 3 D). In brief, 30 mM Ca2+ was applied to one side of the channel and 30 mM Cs+ was applied to the other. The selectivity between Ca2+ and Cs+ can then be calculated from the reversal potential (see METHODS). The reversal potential was near +40 mV (n = 10), indicating the pInsP3R
1-4 pore was Ca2+ selective (PCa/PCs ~ 6.3). Thus, the pInsP3R
1-4 protein forms a high conductance and Ca2+ selective pore.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The principal functional attribute of the InsP3R is its capacity to operate as an intracellular Ca2+ release channel. The permeation and InsP3 regulation of the native type 1 InsP3R pore have been defined in bilayer studies (
The original analysis of the InsP3R cDNA suggested the existence of a channel-forming domain near the COOH terminus of the protein (
Deletion of the sequence bounded by the fifth and sixth TMRs (i.e., the pInsP3R5-6 mutant) did not form detectable Ca2+ channels. However, this mutant protein did occasionally (~15% of attempts) induce a very small, sustained nonspecific leak current. The leak current reversed at 0 mV and no clear opening or closing events were observed. Thus, the leak current was not attributed to the opening and closing of an ion channel. It is more likely that this leak current was due to destabilization of the bilayer after incorporation of integral nonchannel-forming protein. A similar leak current was observed in a previous study with our pInsP3R
T1ALT construct (
T1ALT construct contains all six TMRs. The implication is that the pInsP3R
T1ALT mutant formed a constitutively closed channel, while the pInsP3R
5-6 mutant formed a constitutively open Ca2+ release channel.
Deletion of the first four TMRs (i.e., the pInsP3R1-4 mutant) did form high conductance fast gating ion channels. Control experiments with SS cDNAtransfected cells indicated that the appearance of these channels was not due to some endogenous COS-1 cell protein or factor. The activity of the pInsP3R
1-4 channel was not modified by the addition of InsP3 or heparin. This is interesting because the protein binds InsP3, and this binding is blocked by heparin. In the absence of pharmacological tools, channel identity was thus confirmed by its permeation profile. The pInsP3R
1-4 channel was permeable to both monovalent (i.e., Cs+) and divalent (Ca2+) cations. The Cs+ and Ca2+ conductances were ~280 and 60 pS, respectively. The channel was cation selective, with a Ca2+/Cs+ permeability ratio of 6.3. These values match those described for the wild-type InsP3R channel (
The absence of InsP3 regulation despite InsP3 binding suggests that the deleted sequence (TMRs 14, residues 22112416) may couple binding to channel gating. Alternatively, the missing sequence may annul ligand regulation by sterically limiting pore structure. For example, removing surrounding TMRs could energetically restrict molecular motions in pore structure needed for normal ligand regulation. Such restricted molecular motion could be manifested as a constitutively open pore.
In summary, this study has localized the InsP3R pore to a region of 191 amino acids near the COOH terminus of the protein. This region includes the fifth and sixth TMR and interceding loop. We suggest that a putative leucine zipper may infer the structural integrity needed to form a stable pore. A sequence alignment between the RyR and the InsP3R pore-forming region reveals potential "hot spots" for future mutagenesis studies. These hot spots include the valine/isoleucine residues (i.e., the ß-branched amino acids) in the fifth and sixth TMR and the cluster of conserved glycines in the fifth to sixth TMR loop.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Dr. Thomas Südhof for the kind gift of the InsP3R cDNA.
This work was supported by National Institutes of Health grants R29-MH53367 and R01-HL58851 (G.A. Mignery), and R01-HL570832 (M. Fill). M. Fill is an American Heart Association established investigator.
Submitted: May 17, 1999; Revised: June 23, 1999; Accepted: June 24, 1999.
1used in this paper: ER, endoplasmic reticulum; InsP3R, inositol 1,4,5-trisphosphate receptor; RyR, ryanodine receptor; SS, salmon sperm; TMR, transmembrane region
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baudet, S., Hove-Madsen, L., Bers, D.M. (1994) How to make and use calcium-specific mini- and microelectrodes. Methods Cell Biol. 40:9-114.
Balshaw, D., Gao, L., Meissner, G. (1999) Luminal loop of the ryanodine receptor: a pore-forming segment? Proc. Natl. Acad. Sci. USA 96:3345-3347
Bezprozvanny, I., Watras, J., Ehrlich, B.E. (1991) Bell-shaped calcium-response curves of Ins(1,4,5)P3 and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature. 351:751-754[Medline].
Bezprozvanny, I., Ehrlich, B.E. (1994) InsP3 receptor: functional properties and regulation. In Peracchia C., ed. In Handbook of Membrane Channels. NY, NY, Academic Press, 511-526.
Bezprozvanny, I., Ehrlich, B.E. (1995) The inositol 1,4,5-trisphosphate (InsP3) receptor. J. Membr. Biol 145:205-216[Medline].
De Smedt, H., Missiaen, L., Parys, J.B., Henning, R.H., Sienaert, I., Vanlingen, S., Gijsens, A., Himpens, B., Casteels, R. (1997) Isoform diversity of the inositol 1,4,5-trisphosphate receptor in cell types of mouse origin. Biochem. J. 322:575-583[Medline].
Gao, L., Tripathy, A., Xe, L., Pasek, D., Balshaw, D., Xin, C., Meissner, G. (1999) Mutation of charged amino acids in a putative lumenal loop of the skeletal muscle Ca2+ release channel results in the loss of high affinity ryanodine binding. Biophys. J. 76:A303.
Gorman, C., 1985. DNA Cloning. Vol. II. D.M. Glover, editor. IRL Press, Oxford, UK. 143190.
Hagar, R.E., Burgstahler, A.D., Nathanson, M.H., Ehrlich, B.E. (1998) Type-III InsP3 receptor channel stays open in the presence of increased calcium. Nature. 396:81-84[Medline].
Joseph, S.K. (1996) The inositol trisphosphate receptor family. Cell Signal 8:1-7[Medline].
Joseph, S.K., Boehning, D., Pierson, S., Nicchitta, C.V. (1997) Membrane insertion, glycosylation, and oligomerization of inositol trisphosphate receptors in a cell-free translation system. J.Biol.Chem 272:1579-1588
Kaznacheyeva, E., Lupu, V.D., Bezprozvanny, I. (1998) Single channel properties of inositol trisphosphate receptor heterologously expressed in HEK-293 cells. J. Gen. Physiol. 111:847-856
Lee, K.S., Tsien, R.W. (1982) Reversal of current through Ca2+ channels in dialyzed single heart cells. Nature. 297:498-501[Medline].
Lynch, P.J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J.J.A., Vaughan, P., Zafra, G., MacLennan, D.H., McCarthy, T.V. (1999) A mutation in the transmembrane/lumenal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease. Proc. Natl. Acad. Sci. USA. 96:4164-4169
Michikawa, T., Hamanaka, H., Otsu, H., Yamamoto, A., Miyawaki, A., Furuichi, T., Tashiro, Y., Micoshiba, K. (1994) Transmembrane topology and sites of glycosylation of the inositol trisphosphate receptor. J. Biol. Chem. 269:9184-9189
Mignery, G.A., Südhof, T.C., Takei, K., DeCamilli, P. (1989) Putative receptor for inositol 1,4,5-trisphosphate similar to ryanodine receptor. Nature. 342:192-195[Medline].
Mignery, G.A., Südhof, T.C. (1990) The ligand-binding site and transduction mechanism in the inositol-1,4,5-trisphosphate receptor. EMBO (Eur. Mol. Biol. Organ.) J. 9:3893-3898[Abstract].
Mignery, G.A., Newton, C.L., Archer, B.T., III, Südhof, T.C. (1990) Structure and expression of the rat inositol-1,4,5-trisphosphate receptor. J. Biol. Chem 265:12679-12685
Mignery, G.A., Südhof, T.C. (1993) Molecular analysis of inositol 1,4,5-trisphosphate receptors. Methods Neurosci. 18:247-265.
Nakanishi, S., Fujii, A., Nakade, S., Mikoshiba, K. (1996) Immunohistochemical localization of inositol 1,4,5-trisphosphate receptors in non-neural tissues, with special reference to epithelia, the reproductive system and muscular tissues. Cell Tissue Res. 285:235-251[Medline].
Newton, C.L., Mignery, G.A., Südhof, T.C. (1994) Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J. Biol. Chem 269:28613-28619
Perez, P.J., Ramos-Franco, J., Fill, M., Mignery, G.A. (1997) Identification and functional reconstitution of the type-2 InsP3 receptor from ventricular cardiac myocytes. J. Biol. Chem 272:23961-23969
Ramos-Franco, J., Fill, M., Mignery, G.A. (1998a) Isoform specific function of single inositol 1,4,5-trisphosphate receptor channels. Biophys. J. 75:834-839
Ramos-Franco, J., Fill, M., Mignery, G.A. (1998b) Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants. Biophys. J. 75:2783-2793
Sayers, L.G., Miyawaki, A., Muto, A., Takeshita, H., Yamamoto, A., Michikawa, T., Furuichi, T., Mikoshiba, K. (1997) Intracellular targeting and homotetramer targeting of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the membrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation. Biochem. J. 323:273-280[Medline].
Simmerman, H.K.B., Kobayashi, Y.M., Autry, J.M., Jones, L.R. (1996) A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. J. Biol. Chem 271:5941-5946
Südhof, T.C., Newton, C.L., Archer, B.T., Ushkaryov, Y.A., Mignery, G.A. (1991) The structure of a novel InsP3 receptor. EMBO (Eur. Mol. Biol. Organ.) J. 10:3199-3206[Abstract].
Tinker, A., Williams, A.J. (1992) Divalent cation conduction in the ryanodine receptor of sheep cardiac muscle sarcoplasmic reticulum. J. Gen. Physiol 100:479-493[Abstract].