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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 stabilizes an aberrant inactive gating mode. (A) Diary plots of single channel activity in Kir 1.1a 351X vs. the wild type. Shown are plots of open probability measured in 15-s intervals over 20 min. Single channel recordings were obtained in the cell-attached mode (Vm = −80 mV) from oocytes injected with either wild-type or Kir 1.1a 351X cRNA. The mutant exhibits an inactive mode that has a mean lifetime of 5.12 min. (B) Average open probabilities for both wild-type and truncated mutants (n = 4–5) measured over the duration of a 15–20-min recording (*P < 0.0001). The reduced Po of Kir 1.1a 351X results from the stabilization of the long-lived inactive state.
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Figure 10: COOH-terminal truncation stabilizes an aberrant inactive gating mode. (A) Diary plots of single channel activity in Kir 1.1a 351X vs. the wild type. Shown are plots of open probability measured in 15-s intervals over 20 min. Single channel recordings were obtained in the cell-attached mode (Vm = −80 mV) from oocytes injected with either wild-type or Kir 1.1a 351X cRNA. The mutant exhibits an inactive mode that has a mean lifetime of 5.12 min. (B) Average open probabilities for both wild-type and truncated mutants (n = 4–5) measured over the duration of a 15–20-min recording (*P < 0.0001). The reduced Po of Kir 1.1a 351X results from the stabilization of the long-lived inactive state.

Mentions: By identifying an active channel retaining a defect, Kir 1.1a 351X, the mechanism underlying the role of the COOH-terminal domain could be uncovered. The nature of the defect was elucidated by comparing the single channel properties (cell-attached configuration) of Kir 1.1a 351X (n = 5) to the wild type (n = 4). Aberrant gating behavior was revealed in long duration (∼20 min) recordings, where Po was continuously monitored in 15-s intervals. The results from two representative recordings are shown in Fig. 10 A. In contrast to the sustained high open probability kinetics of the wild-type channel, Kir 1.1a 351X channels exhibited bursts of channel activity, “active gating,” interrupted by sojourns in a long-lived inactive mode (tinactive = 5.12 min). As a consequence of the long-lived inactive state, the open probability of the Kir 1.1a 351X was significantly reduced compared with the wild-type channel (0.36 ± 0.02 vs. 0.91 ± 0.01, respectively; P < 0.0001). During the active gating mode, Kir 1.1a 351X channels exhibited the same single channel conductance (γ = 40 ± 2 pS), open and closed times (τo = 20 ± 1.2 ms; τc = 1.2 ± 0.1 ms) as wild-type channels (Fig. 11), indicating that the COOH-terminal deletion does not alter the conduction pathway. Truncating the extreme COOH terminus of Kir 1.1a reduces channel activity by dramatically increasing the probability of exhibiting an inactive gating mode. This phenotype is unique to Kir 1.1a 351X; the open probability and single channel conductance of Kir 1.1a 361X and Kir 1.1a 366X are not statistically different from the wild-type channel (Fig. 10 B).


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 stabilizes an aberrant inactive gating mode. (A) Diary plots of single channel activity in Kir 1.1a 351X vs. the wild type. Shown are plots of open probability measured in 15-s intervals over 20 min. Single channel recordings were obtained in the cell-attached mode (Vm = −80 mV) from oocytes injected with either wild-type or Kir 1.1a 351X cRNA. The mutant exhibits an inactive mode that has a mean lifetime of 5.12 min. (B) Average open probabilities for both wild-type and truncated mutants (n = 4–5) measured over the duration of a 15–20-min recording (*P < 0.0001). The reduced Po of Kir 1.1a 351X results from the stabilization of the long-lived inactive state.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: COOH-terminal truncation stabilizes an aberrant inactive gating mode. (A) Diary plots of single channel activity in Kir 1.1a 351X vs. the wild type. Shown are plots of open probability measured in 15-s intervals over 20 min. Single channel recordings were obtained in the cell-attached mode (Vm = −80 mV) from oocytes injected with either wild-type or Kir 1.1a 351X cRNA. The mutant exhibits an inactive mode that has a mean lifetime of 5.12 min. (B) Average open probabilities for both wild-type and truncated mutants (n = 4–5) measured over the duration of a 15–20-min recording (*P < 0.0001). The reduced Po of Kir 1.1a 351X results from the stabilization of the long-lived inactive state.
Mentions: By identifying an active channel retaining a defect, Kir 1.1a 351X, the mechanism underlying the role of the COOH-terminal domain could be uncovered. The nature of the defect was elucidated by comparing the single channel properties (cell-attached configuration) of Kir 1.1a 351X (n = 5) to the wild type (n = 4). Aberrant gating behavior was revealed in long duration (∼20 min) recordings, where Po was continuously monitored in 15-s intervals. The results from two representative recordings are shown in Fig. 10 A. In contrast to the sustained high open probability kinetics of the wild-type channel, Kir 1.1a 351X channels exhibited bursts of channel activity, “active gating,” interrupted by sojourns in a long-lived inactive mode (tinactive = 5.12 min). As a consequence of the long-lived inactive state, the open probability of the Kir 1.1a 351X was significantly reduced compared with the wild-type channel (0.36 ± 0.02 vs. 0.91 ± 0.01, respectively; P < 0.0001). During the active gating mode, Kir 1.1a 351X channels exhibited the same single channel conductance (γ = 40 ± 2 pS), open and closed times (τo = 20 ± 1.2 ms; τc = 1.2 ± 0.1 ms) as wild-type channels (Fig. 11), indicating that the COOH-terminal deletion does not alter the conduction pathway. Truncating the extreme COOH terminus of Kir 1.1a reduces channel activity by dramatically increasing the probability of exhibiting an inactive gating mode. This phenotype is unique to Kir 1.1a 351X; the open probability and single channel conductance of Kir 1.1a 361X and Kir 1.1a 366X are not statistically different from the wild-type channel (Fig. 10 B).

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