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Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

Ramos-Franco J, Galvan D, Mignery GA, Fill M - J. Gen. Physiol. (1999)

Bottom Line: These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80).We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore.Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153-5500, USA.

ABSTRACT
The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R 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 InsP(3)R pore. Mutant InsP(3)Rs 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; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.

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Construction and expression of the type 1 (SI−/SII+) InsP3 receptor membrane spanning domain deletion plasmids. (A) Schematic representation of the constructions used in this study. Membrane spanning region deletions pInsP3RΔ5-6 and pInsP3RΔ1-4 are illustrated below the full length receptor (InsP3R-T1). Deleted residues in pInsP3RΔ5-6 and pInsP3RΔ1-4 (residues 2398–2589 and 2211–2416, respectively) are indicated as unshaded regions. Vertical bars represent the membrane spanning domains. (B) The 150 amino acids bounded by the fifth and sixth TMRs of the type 1 InsP3R are aligned with the RyR2 sequence. The fifth and sixth InsP3R TMRs are boxed. Identical residues are shaded. Marked residues indicate identity between all three InsP3R isoforms and RyR2. (C) Western immunoblot of microsomal protein (10 μg, all lanes) from COS-1 cells transiently transfected with control SS DNA, pInsP3RΔ5-6, pInsP3RΔ1-4, and the full-length type 1 receptor (InsP3R-T1). The Western blot was probed with a type 1 specific carboxyl-terminal antipeptide antibody (Ramos-Franco et al. 1998b), and immunoreactive protein was detected using chemiluminescence reagents (Amersham Life Sciences, Inc.). Similar results were observed using a type 1 amino-terminal antibody (data not shown).
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Figure 1: Construction and expression of the type 1 (SI−/SII+) InsP3 receptor membrane spanning domain deletion plasmids. (A) Schematic representation of the constructions used in this study. Membrane spanning region deletions pInsP3RΔ5-6 and pInsP3RΔ1-4 are illustrated below the full length receptor (InsP3R-T1). Deleted residues in pInsP3RΔ5-6 and pInsP3RΔ1-4 (residues 2398–2589 and 2211–2416, respectively) are indicated as unshaded regions. Vertical bars represent the membrane spanning domains. (B) The 150 amino acids bounded by the fifth and sixth TMRs of the type 1 InsP3R are aligned with the RyR2 sequence. The fifth and sixth InsP3R TMRs are boxed. Identical residues are shaded. Marked residues indicate identity between all three InsP3R isoforms and RyR2. (C) Western immunoblot of microsomal protein (10 μg, all lanes) from COS-1 cells transiently transfected with control SS DNA, pInsP3RΔ5-6, pInsP3RΔ1-4, and the full-length type 1 receptor (InsP3R-T1). The Western blot was probed with a type 1 specific carboxyl-terminal antipeptide antibody (Ramos-Franco et al. 1998b), and immunoreactive protein was detected using chemiluminescence reagents (Amersham Life Sciences, Inc.). Similar results were observed using a type 1 amino-terminal antibody (data not shown).

Mentions: The full-length (pInsP3R-T1) and TMR-deletion (pInsP3RΔ1-4 and pInsP3RΔ5-6) mutants were transfected into COS-1 cells using the DEAE-dextran method (Gorman 1985). A schematic of the full-length and deletion mutants used in this study is shown in Fig. 1 A. The putative pore region of the InsP3R includes the 150 amino acids bounded by the fifth and sixth TMRs. A sequence aligned between the type 1 InsP3R and ryanodine receptor 2 (RyR2) proteins over this region is illustrated in Fig. 1 B. The fifth and sixth InsP3R TMRs are boxed, and identical residues in the InsP3R and RyR sequences are shaded. Residues marked by a star indicate those conserved between all three InsP3R isoforms and RyR2. These expression vectors were under the control of the cytomegalovirus promoter (Mignery et al. 1990), and these plasmids expressed immunoreactive InsP3R protein. Microsomes prepared from COS-1 cells transfected with sheared SS DNA revealed no immunoreactive endogenous receptor protein (Fig. 1 C). Extended exposures of the SS DNA Western blots revealed only low levels of immunoreactive protein (data not shown). Microsomes (10 μg protein) from cells expressing the pInsP3R-T1, pInsP3RΔ1-4, and pInsP3RΔ5-6 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 Fig. 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 (Ramos-Franco et al. 1998b). The absence of full-length receptor in the TMR-deletion mutant lanes of the Western blot indicates that there was no substantial upregulation of endogenous type 1 receptor in this study (Fig. 1 C). The abundance of recombinant InsP3R protein (compared with the endogenous receptor) was comparable with that observed in our previous recombinant InsP3R studies (Ramos-Franco et al. 1998b). Thus, proteoliposomes prepared from transfected COS-1 cells contain predominantly recombinant protein. These proteoliposomes can then be reconstituted into planar lipid bilayers to define the single channel properties of the mutant InsP3R channels. This strategy to define the function of recombinant InsP3R channels has been successfully applied by two laboratories (Kaznacheyeva et al. 1998; Ramos-Franco et al. 1998b).


Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

Ramos-Franco J, Galvan D, Mignery GA, Fill M - J. Gen. Physiol. (1999)

Construction and expression of the type 1 (SI−/SII+) InsP3 receptor membrane spanning domain deletion plasmids. (A) Schematic representation of the constructions used in this study. Membrane spanning region deletions pInsP3RΔ5-6 and pInsP3RΔ1-4 are illustrated below the full length receptor (InsP3R-T1). Deleted residues in pInsP3RΔ5-6 and pInsP3RΔ1-4 (residues 2398–2589 and 2211–2416, respectively) are indicated as unshaded regions. Vertical bars represent the membrane spanning domains. (B) The 150 amino acids bounded by the fifth and sixth TMRs of the type 1 InsP3R are aligned with the RyR2 sequence. The fifth and sixth InsP3R TMRs are boxed. Identical residues are shaded. Marked residues indicate identity between all three InsP3R isoforms and RyR2. (C) Western immunoblot of microsomal protein (10 μg, all lanes) from COS-1 cells transiently transfected with control SS DNA, pInsP3RΔ5-6, pInsP3RΔ1-4, and the full-length type 1 receptor (InsP3R-T1). The Western blot was probed with a type 1 specific carboxyl-terminal antipeptide antibody (Ramos-Franco et al. 1998b), and immunoreactive protein was detected using chemiluminescence reagents (Amersham Life Sciences, Inc.). Similar results were observed using a type 1 amino-terminal antibody (data not shown).
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Related In: Results  -  Collection

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Figure 1: Construction and expression of the type 1 (SI−/SII+) InsP3 receptor membrane spanning domain deletion plasmids. (A) Schematic representation of the constructions used in this study. Membrane spanning region deletions pInsP3RΔ5-6 and pInsP3RΔ1-4 are illustrated below the full length receptor (InsP3R-T1). Deleted residues in pInsP3RΔ5-6 and pInsP3RΔ1-4 (residues 2398–2589 and 2211–2416, respectively) are indicated as unshaded regions. Vertical bars represent the membrane spanning domains. (B) The 150 amino acids bounded by the fifth and sixth TMRs of the type 1 InsP3R are aligned with the RyR2 sequence. The fifth and sixth InsP3R TMRs are boxed. Identical residues are shaded. Marked residues indicate identity between all three InsP3R isoforms and RyR2. (C) Western immunoblot of microsomal protein (10 μg, all lanes) from COS-1 cells transiently transfected with control SS DNA, pInsP3RΔ5-6, pInsP3RΔ1-4, and the full-length type 1 receptor (InsP3R-T1). The Western blot was probed with a type 1 specific carboxyl-terminal antipeptide antibody (Ramos-Franco et al. 1998b), and immunoreactive protein was detected using chemiluminescence reagents (Amersham Life Sciences, Inc.). Similar results were observed using a type 1 amino-terminal antibody (data not shown).
Mentions: The full-length (pInsP3R-T1) and TMR-deletion (pInsP3RΔ1-4 and pInsP3RΔ5-6) mutants were transfected into COS-1 cells using the DEAE-dextran method (Gorman 1985). A schematic of the full-length and deletion mutants used in this study is shown in Fig. 1 A. The putative pore region of the InsP3R includes the 150 amino acids bounded by the fifth and sixth TMRs. A sequence aligned between the type 1 InsP3R and ryanodine receptor 2 (RyR2) proteins over this region is illustrated in Fig. 1 B. The fifth and sixth InsP3R TMRs are boxed, and identical residues in the InsP3R and RyR sequences are shaded. Residues marked by a star indicate those conserved between all three InsP3R isoforms and RyR2. These expression vectors were under the control of the cytomegalovirus promoter (Mignery et al. 1990), and these plasmids expressed immunoreactive InsP3R protein. Microsomes prepared from COS-1 cells transfected with sheared SS DNA revealed no immunoreactive endogenous receptor protein (Fig. 1 C). Extended exposures of the SS DNA Western blots revealed only low levels of immunoreactive protein (data not shown). Microsomes (10 μg protein) from cells expressing the pInsP3R-T1, pInsP3RΔ1-4, and pInsP3RΔ5-6 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 Fig. 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 (Ramos-Franco et al. 1998b). The absence of full-length receptor in the TMR-deletion mutant lanes of the Western blot indicates that there was no substantial upregulation of endogenous type 1 receptor in this study (Fig. 1 C). The abundance of recombinant InsP3R protein (compared with the endogenous receptor) was comparable with that observed in our previous recombinant InsP3R studies (Ramos-Franco et al. 1998b). Thus, proteoliposomes prepared from transfected COS-1 cells contain predominantly recombinant protein. These proteoliposomes can then be reconstituted into planar lipid bilayers to define the single channel properties of the mutant InsP3R channels. This strategy to define the function of recombinant InsP3R channels has been successfully applied by two laboratories (Kaznacheyeva et al. 1998; Ramos-Franco et al. 1998b).

Bottom Line: These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80).We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore.Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153-5500, USA.

ABSTRACT
The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R 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 InsP(3)R pore. Mutant InsP(3)Rs 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; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.

Show MeSH