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

Fusion of EGFP to the Kir 1.1a NH2 terminus does not alter channel function. Shown are representative single channel recording (Vm = −80 mV) and corresponding current–voltage relationship, obtained in the cell-attached mode from oocytes injected with EGFP-Kir 1.1a cRNA. EGFP-Kir 1.1a opens with the identical inward slope conductance (bottom) and open probability (0.91 ± 0.01) as the wild-type channel.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230538&req=5

Figure 7: Fusion of EGFP to the Kir 1.1a NH2 terminus does not alter channel function. Shown are representative single channel recording (Vm = −80 mV) and corresponding current–voltage relationship, obtained in the cell-attached mode from oocytes injected with EGFP-Kir 1.1a cRNA. EGFP-Kir 1.1a opens with the identical inward slope conductance (bottom) and open probability (0.91 ± 0.01) as the wild-type channel.

Mentions: To test the role of the COOH-terminal domain (amino acids 332–391) in determining membrane trafficking and plasma membrane stability, NH2-terminal enhanced green fluorescent protein fusion proteins of Kir 1.1a and Kir 1.1a 331X were constructed and expressed in oocytes. This strategy was favored over an antibody binding assay (Firsov et al. 1996) because incorporation of an external epitope (Flag or AUI) abolished Kir 1.1a function. As shown in Fig. 7, NH2-terminal attachment of EGFP had no effect on the single channel conductance (γ = 39 ± 3 pS) or high open probability kinetics of (Po = 0.91 ± 0.01) of Kir 1.1a (n = 4). The lack of EGFP-dependent functional effects offers compelling evidence that channel structure is not altered by the NH2-terminal fusion protein.


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)

Fusion of EGFP to the Kir 1.1a NH2 terminus does not alter channel function. Shown are representative single channel recording (Vm = −80 mV) and corresponding current–voltage relationship, obtained in the cell-attached mode from oocytes injected with EGFP-Kir 1.1a cRNA. EGFP-Kir 1.1a opens with the identical inward slope conductance (bottom) and open probability (0.91 ± 0.01) as the wild-type channel.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Fusion of EGFP to the Kir 1.1a NH2 terminus does not alter channel function. Shown are representative single channel recording (Vm = −80 mV) and corresponding current–voltage relationship, obtained in the cell-attached mode from oocytes injected with EGFP-Kir 1.1a cRNA. EGFP-Kir 1.1a opens with the identical inward slope conductance (bottom) and open probability (0.91 ± 0.01) as the wild-type channel.
Mentions: To test the role of the COOH-terminal domain (amino acids 332–391) in determining membrane trafficking and plasma membrane stability, NH2-terminal enhanced green fluorescent protein fusion proteins of Kir 1.1a and Kir 1.1a 331X were constructed and expressed in oocytes. This strategy was favored over an antibody binding assay (Firsov et al. 1996) because incorporation of an external epitope (Flag or AUI) abolished Kir 1.1a function. As shown in Fig. 7, NH2-terminal attachment of EGFP had no effect on the single channel conductance (γ = 39 ± 3 pS) or high open probability kinetics of (Po = 0.91 ± 0.01) of Kir 1.1a (n = 4). The lack of EGFP-dependent functional effects offers compelling evidence that channel structure is not altered by the NH2-terminal fusion protein.

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