Limits...
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.

Show MeSH

Related in: MedlinePlus

The conductance and selectivity of the pInsP3RΔ1-4 pore. (A) Sample single channel activity at three different voltages (0, 20, and 40 mV) recorded with Cs+ as a charge carrier. The open and closed current levels are indicated. Calibration bars represent 1 s and 20 pA. (B) Current–voltage relationship in asymmetrical (220/20 mM) CsCH3SO3 solutions. Each point represents mean (±SEM) of several (three to nine) determinations on different channels. Points without error bars had error levels within the area of the symbol. (C) Current–voltage relationship in asymmetrical Ca2+ solutions [0/30 mM Ca(CH3SO3)2]. Each point represents mean (±SEM) of several (three to seven) determinations on different channels. (D) Current–voltage relationship measured under biionic conditions (30 mM Ca2+ trans, 30 mM Cs+ cis). Each point represents mean (±SEM) of several (4–10) determinations on different channels. Arrow indicates the extrapolated reversal potential.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230642&req=5

Figure 3: The conductance and selectivity of the pInsP3RΔ1-4 pore. (A) Sample single channel activity at three different voltages (0, 20, and 40 mV) recorded with Cs+ as a charge carrier. The open and closed current levels are indicated. Calibration bars represent 1 s and 20 pA. (B) Current–voltage relationship in asymmetrical (220/20 mM) CsCH3SO3 solutions. Each point represents mean (±SEM) of several (three to nine) determinations on different channels. Points without error bars had error levels within the area of the symbol. (C) Current–voltage relationship in asymmetrical Ca2+ solutions [0/30 mM Ca(CH3SO3)2]. Each point represents mean (±SEM) of several (three to seven) determinations on different channels. (D) Current–voltage relationship measured under biionic conditions (30 mM Ca2+ trans, 30 mM Cs+ cis). Each point represents mean (±SEM) of several (4–10) determinations on different channels. Arrow indicates the extrapolated reversal potential.

Mentions: The permeation properties of the pInsP3RΔ1-4 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 Fig. 3 A. A sustained high Po was a fundamental feature the pInsP3RΔ1-4 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.


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)

The conductance and selectivity of the pInsP3RΔ1-4 pore. (A) Sample single channel activity at three different voltages (0, 20, and 40 mV) recorded with Cs+ as a charge carrier. The open and closed current levels are indicated. Calibration bars represent 1 s and 20 pA. (B) Current–voltage relationship in asymmetrical (220/20 mM) CsCH3SO3 solutions. Each point represents mean (±SEM) of several (three to nine) determinations on different channels. Points without error bars had error levels within the area of the symbol. (C) Current–voltage relationship in asymmetrical Ca2+ solutions [0/30 mM Ca(CH3SO3)2]. Each point represents mean (±SEM) of several (three to seven) determinations on different channels. (D) Current–voltage relationship measured under biionic conditions (30 mM Ca2+ trans, 30 mM Cs+ cis). Each point represents mean (±SEM) of several (4–10) determinations on different channels. Arrow indicates the extrapolated reversal potential.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: The conductance and selectivity of the pInsP3RΔ1-4 pore. (A) Sample single channel activity at three different voltages (0, 20, and 40 mV) recorded with Cs+ as a charge carrier. The open and closed current levels are indicated. Calibration bars represent 1 s and 20 pA. (B) Current–voltage relationship in asymmetrical (220/20 mM) CsCH3SO3 solutions. Each point represents mean (±SEM) of several (three to nine) determinations on different channels. Points without error bars had error levels within the area of the symbol. (C) Current–voltage relationship in asymmetrical Ca2+ solutions [0/30 mM Ca(CH3SO3)2]. Each point represents mean (±SEM) of several (three to seven) determinations on different channels. (D) Current–voltage relationship measured under biionic conditions (30 mM Ca2+ trans, 30 mM Cs+ cis). Each point represents mean (±SEM) of several (4–10) determinations on different channels. Arrow indicates the extrapolated reversal potential.
Mentions: The permeation properties of the pInsP3RΔ1-4 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 Fig. 3 A. A sustained high Po was a fundamental feature the pInsP3RΔ1-4 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.

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