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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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Incorporation of single Kir 1.1a 331X mutant at either the NH2- or COOH-terminal position of a tandem tetramer suppresses channel activity. (A) Representative families of whole-cell currents, (B) composite current–voltage relationships, and (C) normalized macroscopic Ba2+-sensitive currents (Vm = −90 mV) measured from oocytes injected with equivalent amounts of 4wt, 3wt + 1mut, or 1mut + 3wt concatenated tetramer cRNA (1 ng; *P < 0.0001) are shown.
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Figure 5: Incorporation of single Kir 1.1a 331X mutant at either the NH2- or COOH-terminal position of a tandem tetramer suppresses channel activity. (A) Representative families of whole-cell currents, (B) composite current–voltage relationships, and (C) normalized macroscopic Ba2+-sensitive currents (Vm = −90 mV) measured from oocytes injected with equivalent amounts of 4wt, 3wt + 1mut, or 1mut + 3wt concatenated tetramer cRNA (1 ng; *P < 0.0001) are shown.

Mentions: Having demonstrated that concatenation of subunits does not affect channel function, we tested the functional effects of Kir 1.1a 331X within a tandem tetramer. Incorporation of a single mutant within the concatemeric tetramer, at either the NH2- or COOH-terminal positions (1mut + 3wt or 3wt + 1mut, respectively) completely suppressed macroscopic channel activity. These results are summarized in Fig. 5. In contrast to the 4wt concatemer (−2.56 ± 0.34 μA, n = 25), the 1mut + 3wt and 3wt + 1mut concatemers exhibited no measurable currents above background (−0.15 ± 0.03 and −0.10 ± 0.02 μA, respectively, n = 12, P < 0.0001). The complete dominant negative effect was confirmed at the single channel level. Except for occasional endogenous stretch-activated channel openings in some patches, no significant activity could be detected in on-cell patches of oocytes injected with either mutant-containing concatemer (Fig. 6, Fig. 3wt + 1mut, n = 14, total recording time = 56 min; 1mut + 3wt, n = 17, total recording time = 31 min).


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)

Incorporation of single Kir 1.1a 331X mutant at either the NH2- or COOH-terminal position of a tandem tetramer suppresses channel activity. (A) Representative families of whole-cell currents, (B) composite current–voltage relationships, and (C) normalized macroscopic Ba2+-sensitive currents (Vm = −90 mV) measured from oocytes injected with equivalent amounts of 4wt, 3wt + 1mut, or 1mut + 3wt concatenated tetramer cRNA (1 ng; *P < 0.0001) are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Incorporation of single Kir 1.1a 331X mutant at either the NH2- or COOH-terminal position of a tandem tetramer suppresses channel activity. (A) Representative families of whole-cell currents, (B) composite current–voltage relationships, and (C) normalized macroscopic Ba2+-sensitive currents (Vm = −90 mV) measured from oocytes injected with equivalent amounts of 4wt, 3wt + 1mut, or 1mut + 3wt concatenated tetramer cRNA (1 ng; *P < 0.0001) are shown.
Mentions: Having demonstrated that concatenation of subunits does not affect channel function, we tested the functional effects of Kir 1.1a 331X within a tandem tetramer. Incorporation of a single mutant within the concatemeric tetramer, at either the NH2- or COOH-terminal positions (1mut + 3wt or 3wt + 1mut, respectively) completely suppressed macroscopic channel activity. These results are summarized in Fig. 5. In contrast to the 4wt concatemer (−2.56 ± 0.34 μA, n = 25), the 1mut + 3wt and 3wt + 1mut concatemers exhibited no measurable currents above background (−0.15 ± 0.03 and −0.10 ± 0.02 μA, respectively, n = 12, P < 0.0001). The complete dominant negative effect was confirmed at the single channel level. Except for occasional endogenous stretch-activated channel openings in some patches, no significant activity could be detected in on-cell patches of oocytes injected with either mutant-containing concatemer (Fig. 6, Fig. 3wt + 1mut, n = 14, total recording time = 56 min; 1mut + 3wt, n = 17, total recording time = 31 min).

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