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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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A series of truncated mutants delimit amino acids 332–351 as the COOH-terminal domain necessary to maintain channel function. (A) Kir 1.1a channels were reconstructed by adding residues back to the Bartter's mutant, Kir 1.1a 331X. Amino acids 332–361 represent the most conserved domain (30% identity) within the extreme COOH terminus of inward rectifying K+ channels. (B) Shown are normalized Ba2+-sensitive whole cell currents (Vm = βˆ’90 mV) obtained from oocytes expressing each of the truncated mutants depicted in A (*P < 0.01). Replacement of amino acids 332–351 (Kir 1.1a 351X) restored channel activity, while deletion of this domain (Kir 1.1a Ξ”332-351) abolished macroscopic currents.
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Figure 9: A series of truncated mutants delimit amino acids 332–351 as the COOH-terminal domain necessary to maintain channel function. (A) Kir 1.1a channels were reconstructed by adding residues back to the Bartter's mutant, Kir 1.1a 331X. Amino acids 332–361 represent the most conserved domain (30% identity) within the extreme COOH terminus of inward rectifying K+ channels. (B) Shown are normalized Ba2+-sensitive whole cell currents (Vm = βˆ’90 mV) obtained from oocytes expressing each of the truncated mutants depicted in A (*P < 0.01). Replacement of amino acids 332–351 (Kir 1.1a 351X) restored channel activity, while deletion of this domain (Kir 1.1a Ξ”332-351) abolished macroscopic currents.

Mentions: Having found that the extreme COOH terminus is involved in maintaining channel activity, we delimited the minimal domain that is required for functional expression in the hopes of revealing the mechanism underlying the Kir 1.1a 331X defect. In these studies, the channel was gradually reconstructed by sequentially adding portions of the extreme COOH terminus back to Kir 1.1a 331X (Fig. 9 A). Addition of amino acids 332–351 was sufficient to rescue minimal channel function, while deletion of this domain in isolation (Kir 1.1a Ξ”332–351) resulted in the loss of channel function. The results of these studies are summarized in Fig. 9 B. Oocytes injected with either Kir 1.1a 341X or Kir 1.1a Ξ”332–351, like Kir 1.1a 331X, exhibited no Ba2+-sensitive currents above background (n = 6–11). In contrast, Kir 1.1a 351X–injected oocytes displayed significant weakly inward-rectifying, Ba2+-sensitive macroscopic currents (n = 10, P < 0.001). Current density increased further with the addition of 10 more residues (Kir 1.1a 361X, n = 11, P < 0.0001). The addition of five more residues (Kir 1.1a 366X, n = 10) failed to produce a further significant increase in channel activity. Collectively, these studies define amino acids 332–351 as the critical COOH-terminal domain within the extreme COOH terminus that is absolutely required for maintaining channel activity.


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)

A series of truncated mutants delimit amino acids 332–351 as the COOH-terminal domain necessary to maintain channel function. (A) Kir 1.1a channels were reconstructed by adding residues back to the Bartter's mutant, Kir 1.1a 331X. Amino acids 332–361 represent the most conserved domain (30% identity) within the extreme COOH terminus of inward rectifying K+ channels. (B) Shown are normalized Ba2+-sensitive whole cell currents (Vm = βˆ’90 mV) obtained from oocytes expressing each of the truncated mutants depicted in A (*P < 0.01). Replacement of amino acids 332–351 (Kir 1.1a 351X) restored channel activity, while deletion of this domain (Kir 1.1a Ξ”332-351) abolished macroscopic currents.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230538&req=5

Figure 9: A series of truncated mutants delimit amino acids 332–351 as the COOH-terminal domain necessary to maintain channel function. (A) Kir 1.1a channels were reconstructed by adding residues back to the Bartter's mutant, Kir 1.1a 331X. Amino acids 332–361 represent the most conserved domain (30% identity) within the extreme COOH terminus of inward rectifying K+ channels. (B) Shown are normalized Ba2+-sensitive whole cell currents (Vm = βˆ’90 mV) obtained from oocytes expressing each of the truncated mutants depicted in A (*P < 0.01). Replacement of amino acids 332–351 (Kir 1.1a 351X) restored channel activity, while deletion of this domain (Kir 1.1a Ξ”332-351) abolished macroscopic currents.
Mentions: Having found that the extreme COOH terminus is involved in maintaining channel activity, we delimited the minimal domain that is required for functional expression in the hopes of revealing the mechanism underlying the Kir 1.1a 331X defect. In these studies, the channel was gradually reconstructed by sequentially adding portions of the extreme COOH terminus back to Kir 1.1a 331X (Fig. 9 A). Addition of amino acids 332–351 was sufficient to rescue minimal channel function, while deletion of this domain in isolation (Kir 1.1a Ξ”332–351) resulted in the loss of channel function. The results of these studies are summarized in Fig. 9 B. Oocytes injected with either Kir 1.1a 341X or Kir 1.1a Ξ”332–351, like Kir 1.1a 331X, exhibited no Ba2+-sensitive currents above background (n = 6–11). In contrast, Kir 1.1a 351X–injected oocytes displayed significant weakly inward-rectifying, Ba2+-sensitive macroscopic currents (n = 10, P < 0.001). Current density increased further with the addition of 10 more residues (Kir 1.1a 361X, n = 11, P < 0.0001). The addition of five more residues (Kir 1.1a 366X, n = 10) failed to produce a further significant increase in channel activity. Collectively, these studies define amino acids 332–351 as the critical COOH-terminal domain within the extreme COOH terminus that is absolutely required for maintaining channel activity.

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