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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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Single channel currents through the pore formed by the InsP3R TMR-deletion mutant pInsP3RΔ1-4. This deletion mutant is missing the first through fourth TMRs. Representative single channel records obtained with 220 mM CsCH3SO3 as the current carrier. The membrane potential was 0 mV. The open and closed current levels are indicated. The data shown are representative of data collected on six different single channels. Single channel activity monitored in the presence (A) and absence (B) of InsP3, and in the presence of heparin (C). The associated amplitude histograms were obtained from several minutes of recording under each experimental condition. Solid lines represent the best fits of the two Gaussian distributions. Note the consistently high Po and similar unitary currents under all experimental conditions.
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Figure 2: Single channel currents through the pore formed by the InsP3R TMR-deletion mutant pInsP3RΔ1-4. This deletion mutant is missing the first through fourth TMRs. Representative single channel records obtained with 220 mM CsCH3SO3 as the current carrier. The membrane potential was 0 mV. The open and closed current levels are indicated. The data shown are representative of data collected on six different single channels. Single channel activity monitored in the presence (A) and absence (B) of InsP3, and in the presence of heparin (C). The associated amplitude histograms were obtained from several minutes of recording under each experimental condition. Solid lines represent the best fits of the two Gaussian distributions. Note the consistently high Po and similar unitary currents under all experimental conditions.

Mentions: 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 pInsP3RΔ5-6–transfected cells. Incorporation of proteoliposomes containing the pInsP3RΔ1-4 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 pInsP3RΔ1-4 channel is shown in Fig. 2. The pInsP3RΔ1-4 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 (Fig. 2 A) and absence (Fig. 2 B) of InsP3. Single channel activity was also not impacted by the addition of 10 μM ryanodine or 50 μg/ml heparin (Fig. 2 C). Corresponding total amplitude histograms under each experimental condition are also presented in Fig. 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 pInsP3RΔ1-4 protein was not modulated by agents (i.e., InsP3 and heparin) that modulate function of wild-type InsP3R channels. Additionally, the pInsP3RΔ1-4 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 (Bezprozvanny et al. 1991; Ramos-Franco et al. 1998a,Ramos-Franco et al. 1998b). The high Po and absence of channel regulation by InsP3 and heparin indicates that the channel activity observed is not due to endogenous InsP3R channels. The absence of ryanodine action indicates that it is not due to endogenous RyR channels.


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)

Single channel currents through the pore formed by the InsP3R TMR-deletion mutant pInsP3RΔ1-4. This deletion mutant is missing the first through fourth TMRs. Representative single channel records obtained with 220 mM CsCH3SO3 as the current carrier. The membrane potential was 0 mV. The open and closed current levels are indicated. The data shown are representative of data collected on six different single channels. Single channel activity monitored in the presence (A) and absence (B) of InsP3, and in the presence of heparin (C). The associated amplitude histograms were obtained from several minutes of recording under each experimental condition. Solid lines represent the best fits of the two Gaussian distributions. Note the consistently high Po and similar unitary currents under all experimental conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230642&req=5

Figure 2: Single channel currents through the pore formed by the InsP3R TMR-deletion mutant pInsP3RΔ1-4. This deletion mutant is missing the first through fourth TMRs. Representative single channel records obtained with 220 mM CsCH3SO3 as the current carrier. The membrane potential was 0 mV. The open and closed current levels are indicated. The data shown are representative of data collected on six different single channels. Single channel activity monitored in the presence (A) and absence (B) of InsP3, and in the presence of heparin (C). The associated amplitude histograms were obtained from several minutes of recording under each experimental condition. Solid lines represent the best fits of the two Gaussian distributions. Note the consistently high Po and similar unitary currents under all experimental conditions.
Mentions: 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 pInsP3RΔ5-6–transfected cells. Incorporation of proteoliposomes containing the pInsP3RΔ1-4 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 pInsP3RΔ1-4 channel is shown in Fig. 2. The pInsP3RΔ1-4 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 (Fig. 2 A) and absence (Fig. 2 B) of InsP3. Single channel activity was also not impacted by the addition of 10 μM ryanodine or 50 μg/ml heparin (Fig. 2 C). Corresponding total amplitude histograms under each experimental condition are also presented in Fig. 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 pInsP3RΔ1-4 protein was not modulated by agents (i.e., InsP3 and heparin) that modulate function of wild-type InsP3R channels. Additionally, the pInsP3RΔ1-4 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 (Bezprozvanny et al. 1991; Ramos-Franco et al. 1998a,Ramos-Franco et al. 1998b). The high Po and absence of channel regulation by InsP3 and heparin indicates that the channel activity observed is not due to endogenous InsP3R channels. The absence of ryanodine action indicates that it is not due to endogenous RyR channels.

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
Related in: MedlinePlus