Limits...
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.

Show MeSH

Related in: MedlinePlus

Deletion of the extreme COOH terminus does not cause a global structural mutation or prevent subunit oligomerization. (A) Representative families of whole cell currents recorded from oocytes injected with either Kir 1.1a (250 pg) or Kir 1.1a and Kir 1.1a 331X (250 pg each) cRNA. (B) Ba2+-sensitive macroscopic currents (Vm = −90 mV) measured in oocytes coinjected with equivalent amounts of cRNA encoding Kir 1.1a and Kir 1.1 331X or Kir 1.1a and Kir 3.1-AAA (250 pg each), normalized to the mean current of the control group injected with Kir 1.1a alone (250 pg) (*P < 0.005). Kir 3.1-AAA is a mutant, nonconductive form of a G protein–gated Kir channel that exerts a dominant negative effect on Kir 3.1, but does not oligomerize with Kir 1.1a.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230538&req=5

Figure 2: Deletion of the extreme COOH terminus does not cause a global structural mutation or prevent subunit oligomerization. (A) Representative families of whole cell currents recorded from oocytes injected with either Kir 1.1a (250 pg) or Kir 1.1a and Kir 1.1a 331X (250 pg each) cRNA. (B) Ba2+-sensitive macroscopic currents (Vm = −90 mV) measured in oocytes coinjected with equivalent amounts of cRNA encoding Kir 1.1a and Kir 1.1 331X or Kir 1.1a and Kir 3.1-AAA (250 pg each), normalized to the mean current of the control group injected with Kir 1.1a alone (250 pg) (*P < 0.005). Kir 3.1-AAA is a mutant, nonconductive form of a G protein–gated Kir channel that exerts a dominant negative effect on Kir 3.1, but does not oligomerize with Kir 1.1a.

Mentions: An oligomerization defect or global structural mutation can be easily tested by coexpressing the mutant with the wild-type channel. If the mutant protein is synthesized, folded correctly, and capable of oligomerization, the macroscopic current in oocytes coinjected with wild-type and mutant cRNA is predicted to be less than oocytes injected with wild-type alone. Fig. 2 B summarizes the results of such a study. Coexpression of Kir 1.1a 331X with the wild-type channel reduced Ba2+-sensitive macroscopic current by 62 ± 5%, demonstrating that the mutant is capable of exerting a negative influence on the wild-type channel (n = 12, Kir 1.1a 331X; n = 18, Kir 1.1a; P < 0.005).


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)

Deletion of the extreme COOH terminus does not cause a global structural mutation or prevent subunit oligomerization. (A) Representative families of whole cell currents recorded from oocytes injected with either Kir 1.1a (250 pg) or Kir 1.1a and Kir 1.1a 331X (250 pg each) cRNA. (B) Ba2+-sensitive macroscopic currents (Vm = −90 mV) measured in oocytes coinjected with equivalent amounts of cRNA encoding Kir 1.1a and Kir 1.1 331X or Kir 1.1a and Kir 3.1-AAA (250 pg each), normalized to the mean current of the control group injected with Kir 1.1a alone (250 pg) (*P < 0.005). Kir 3.1-AAA is a mutant, nonconductive form of a G protein–gated Kir channel that exerts a dominant negative effect on Kir 3.1, but does not oligomerize with Kir 1.1a.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Deletion of the extreme COOH terminus does not cause a global structural mutation or prevent subunit oligomerization. (A) Representative families of whole cell currents recorded from oocytes injected with either Kir 1.1a (250 pg) or Kir 1.1a and Kir 1.1a 331X (250 pg each) cRNA. (B) Ba2+-sensitive macroscopic currents (Vm = −90 mV) measured in oocytes coinjected with equivalent amounts of cRNA encoding Kir 1.1a and Kir 1.1 331X or Kir 1.1a and Kir 3.1-AAA (250 pg each), normalized to the mean current of the control group injected with Kir 1.1a alone (250 pg) (*P < 0.005). Kir 3.1-AAA is a mutant, nonconductive form of a G protein–gated Kir channel that exerts a dominant negative effect on Kir 3.1, but does not oligomerize with Kir 1.1a.
Mentions: An oligomerization defect or global structural mutation can be easily tested by coexpressing the mutant with the wild-type channel. If the mutant protein is synthesized, folded correctly, and capable of oligomerization, the macroscopic current in oocytes coinjected with wild-type and mutant cRNA is predicted to be less than oocytes injected with wild-type alone. Fig. 2 B summarizes the results of such a study. Coexpression of Kir 1.1a 331X with the wild-type channel reduced Ba2+-sensitive macroscopic current by 62 ± 5%, demonstrating that the mutant is capable of exerting a negative influence on the wild-type channel (n = 12, Kir 1.1a 331X; n = 18, Kir 1.1a; P < 0.005).

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