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A mutation linked with Bartter's syndrome locks Kir 1.1a (ROMK1) channels in a closed state.

Flagg TP, Tate M, Merot J, Welling PA - J. Gen. Physiol. (1999)

Bottom Line: When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization.A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization.Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

ABSTRACT
Mutations in the inward rectifying renal K(+) channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332-391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP-wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332-351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

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COOH-terminal truncation does not alter Kir 1.1a trafficking or plasma membrane stability in the Xenopus oocyte expression system. (A) Representative optical sections acquired from oocytes at a focal plane near the equator using laser-scanning confocal microscopy. Oocytes injected with EGFP, EGFP-Kir 1.1a, or EGFP-Kir 1.1a 331X cRNA were compared with uninjected oocytes. (B) Circumferential fluorescence intensity plotted as a function of the fusion protein cRNA dose injected. No significant differences in circumferential fluorescence distribution and intensity were detected at any injection dose. The solid line represents the linear regression fit of the average circumferential fluorescence for oocytes injected with either wild-type or mutant fusion protein cRNA. C) Macroscopic conductance was measured in oocytes injected with either fusion construct at several cRNA injection doses and is plotted as a function of plasma membrane delimited fluorescence. Slope conductance was determined between −110 and −30 mV from oocytes bathed in 5 mM [K+]o. No conductance above background was detected in oocytes expressing EGFP-Kir 1.1a 331X, consistent with the premise that Kir 1.1a 331X resides in the plasma membrane but is in a nonconductive or inactive conformation.
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Figure 8: COOH-terminal truncation does not alter Kir 1.1a trafficking or plasma membrane stability in the Xenopus oocyte expression system. (A) Representative optical sections acquired from oocytes at a focal plane near the equator using laser-scanning confocal microscopy. Oocytes injected with EGFP, EGFP-Kir 1.1a, or EGFP-Kir 1.1a 331X cRNA were compared with uninjected oocytes. (B) Circumferential fluorescence intensity plotted as a function of the fusion protein cRNA dose injected. No significant differences in circumferential fluorescence distribution and intensity were detected at any injection dose. The solid line represents the linear regression fit of the average circumferential fluorescence for oocytes injected with either wild-type or mutant fusion protein cRNA. C) Macroscopic conductance was measured in oocytes injected with either fusion construct at several cRNA injection doses and is plotted as a function of plasma membrane delimited fluorescence. Slope conductance was determined between −110 and −30 mV from oocytes bathed in 5 mM [K+]o. No conductance above background was detected in oocytes expressing EGFP-Kir 1.1a 331X, consistent with the premise that Kir 1.1a 331X resides in the plasma membrane but is in a nonconductive or inactive conformation.

Mentions: As shown in Fig. 8, whole, unfixed oocytes injected with EGFP fusion protein cRNA were examined using laser scanning confocal microscopy. Uninjected oocytes exhibited a low basal autofluorescence. EGFP was widely distributed throughout the cytoplasm (n = 6). In contrast, both EGFP-Kir 1.1a and EGFP-Kir 1.1a 331X were localized along a distinct zone circumscribing the oocyte (n = 6 in each injection dose). The circumferential fluorescence pattern was maintained in sequential z-plane optical sections through the entire oocyte, consistent with predominant plasma membrane expression. Fluorescence intensity was proportional to the amount of fusion protein cRNA injected, and no significant difference could be detected between EGFP-Kir 1.1a and EGFP-Kir 1.1a 331X at any injection dose (Fig. 8 B). These data support the premise that both channels are expressed on the plasma membrane with equal efficiency.


A mutation linked with Bartter's syndrome locks Kir 1.1a (ROMK1) channels in a closed state.

Flagg TP, Tate M, Merot J, Welling PA - J. Gen. Physiol. (1999)

COOH-terminal truncation does not alter Kir 1.1a trafficking or plasma membrane stability in the Xenopus oocyte expression system. (A) Representative optical sections acquired from oocytes at a focal plane near the equator using laser-scanning confocal microscopy. Oocytes injected with EGFP, EGFP-Kir 1.1a, or EGFP-Kir 1.1a 331X cRNA were compared with uninjected oocytes. (B) Circumferential fluorescence intensity plotted as a function of the fusion protein cRNA dose injected. No significant differences in circumferential fluorescence distribution and intensity were detected at any injection dose. The solid line represents the linear regression fit of the average circumferential fluorescence for oocytes injected with either wild-type or mutant fusion protein cRNA. C) Macroscopic conductance was measured in oocytes injected with either fusion construct at several cRNA injection doses and is plotted as a function of plasma membrane delimited fluorescence. Slope conductance was determined between −110 and −30 mV from oocytes bathed in 5 mM [K+]o. No conductance above background was detected in oocytes expressing EGFP-Kir 1.1a 331X, consistent with the premise that Kir 1.1a 331X resides in the plasma membrane but is in a nonconductive or inactive conformation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230538&req=5

Figure 8: COOH-terminal truncation does not alter Kir 1.1a trafficking or plasma membrane stability in the Xenopus oocyte expression system. (A) Representative optical sections acquired from oocytes at a focal plane near the equator using laser-scanning confocal microscopy. Oocytes injected with EGFP, EGFP-Kir 1.1a, or EGFP-Kir 1.1a 331X cRNA were compared with uninjected oocytes. (B) Circumferential fluorescence intensity plotted as a function of the fusion protein cRNA dose injected. No significant differences in circumferential fluorescence distribution and intensity were detected at any injection dose. The solid line represents the linear regression fit of the average circumferential fluorescence for oocytes injected with either wild-type or mutant fusion protein cRNA. C) Macroscopic conductance was measured in oocytes injected with either fusion construct at several cRNA injection doses and is plotted as a function of plasma membrane delimited fluorescence. Slope conductance was determined between −110 and −30 mV from oocytes bathed in 5 mM [K+]o. No conductance above background was detected in oocytes expressing EGFP-Kir 1.1a 331X, consistent with the premise that Kir 1.1a 331X resides in the plasma membrane but is in a nonconductive or inactive conformation.
Mentions: As shown in Fig. 8, whole, unfixed oocytes injected with EGFP fusion protein cRNA were examined using laser scanning confocal microscopy. Uninjected oocytes exhibited a low basal autofluorescence. EGFP was widely distributed throughout the cytoplasm (n = 6). In contrast, both EGFP-Kir 1.1a and EGFP-Kir 1.1a 331X were localized along a distinct zone circumscribing the oocyte (n = 6 in each injection dose). The circumferential fluorescence pattern was maintained in sequential z-plane optical sections through the entire oocyte, consistent with predominant plasma membrane expression. Fluorescence intensity was proportional to the amount of fusion protein cRNA injected, and no significant difference could be detected between EGFP-Kir 1.1a and EGFP-Kir 1.1a 331X at any injection dose (Fig. 8 B). These data support the premise that both channels are expressed on the plasma membrane with equal efficiency.

Bottom Line: When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization.A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization.Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

ABSTRACT
Mutations in the inward rectifying renal K(+) channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332-391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP-wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332-351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

Show MeSH
Related in: MedlinePlus