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

Dominant negative effects of Kir 1.1a 331X on wild-type Kir 1.1a. Ba2+-sensitive inward current is plotted (I/Io, Vm = −90 mV) as a function of Fmut, the mutant fraction of the total cRNA injected [Fmut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)]. Fmut was adjusted by coinjecting variable amounts of Kir 1.1a 331X with a constant dose of Kir 1.1a (250 pg), and Io is the mean current when Fmut = 0. The dotted line (A) represents the predicted relationship if inclusion of one or more mutant subunits within a tetramer inhibits channel function [I/Io = (1 Fmut)4]. The dashed curve (B) is predicted if two or more subunits are required [I/Io = (1 − Fmut)4 + 4(1 − Fmut)3Fmut]. The data are best fit by an intermediate model, I/Io = (1 − 0.6 Fmut)4, represented by the solid line (C). The coefficient, k, is less than unity, indicating that a single mutant subunit partially inhibits current or that Kir 1.1a331X oligomerizes with a reduced efficiency.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230538&req=5

Figure 3: Dominant negative effects of Kir 1.1a 331X on wild-type Kir 1.1a. Ba2+-sensitive inward current is plotted (I/Io, Vm = −90 mV) as a function of Fmut, the mutant fraction of the total cRNA injected [Fmut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)]. Fmut was adjusted by coinjecting variable amounts of Kir 1.1a 331X with a constant dose of Kir 1.1a (250 pg), and Io is the mean current when Fmut = 0. The dotted line (A) represents the predicted relationship if inclusion of one or more mutant subunits within a tetramer inhibits channel function [I/Io = (1 Fmut)4]. The dashed curve (B) is predicted if two or more subunits are required [I/Io = (1 − Fmut)4 + 4(1 − Fmut)3Fmut]. The data are best fit by an intermediate model, I/Io = (1 − 0.6 Fmut)4, represented by the solid line (C). The coefficient, k, is less than unity, indicating that a single mutant subunit partially inhibits current or that Kir 1.1a331X oligomerizes with a reduced efficiency.

Mentions: To gain further insight into the dominant negative mechanism, increasing doses of mutant cRNA were coinjected with a constant amount of wild-type transcript (Fig. 3; n = 5–14 each dose). Because Kir 1.1a channels are known to be tetrameric in structure (Glowatzki et al. 1995), coinjection should produce five discrete populations, containing zero to four mutant subunits. Assuming random assembly, a binomial probability distribution defined by two parameters, Fmut and n, determines the probability of obtaining each channel population. Fmut, the mutant fraction of the total cRNA injected [Fmut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)], is the probability of incorporating a Kir 1.1a 331X subunit. For a tetrameric channel, the number of subunits, n, is equal to 4. The relative frequency of functional populations determines the resultant macroscopic current density. The predicted relationships for two specific dominant negative models are shown in Fig. 3. Consider the situation when incorporation of one or more mutant subunits abolishes channel activity. The relative current is determined by the probability of assembling four wild-type subunits into a tetramer. In this case, the macroscopic current can be predicted by the relationship, I/Io = (1 − Fmut)4 (Fig. 3, line A). If two or more mutants must be incorporated within a tetramer to inhibit channel activity, an additional term, the probability of forming a channel with one mutant and three wild-type subunits [4 Fmut(1 − Fmut)3], is added to the equation above (line 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)

Dominant negative effects of Kir 1.1a 331X on wild-type Kir 1.1a. Ba2+-sensitive inward current is plotted (I/Io, Vm = −90 mV) as a function of Fmut, the mutant fraction of the total cRNA injected [Fmut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)]. Fmut was adjusted by coinjecting variable amounts of Kir 1.1a 331X with a constant dose of Kir 1.1a (250 pg), and Io is the mean current when Fmut = 0. The dotted line (A) represents the predicted relationship if inclusion of one or more mutant subunits within a tetramer inhibits channel function [I/Io = (1 Fmut)4]. The dashed curve (B) is predicted if two or more subunits are required [I/Io = (1 − Fmut)4 + 4(1 − Fmut)3Fmut]. The data are best fit by an intermediate model, I/Io = (1 − 0.6 Fmut)4, represented by the solid line (C). The coefficient, k, is less than unity, indicating that a single mutant subunit partially inhibits current or that Kir 1.1a331X oligomerizes with a reduced efficiency.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Dominant negative effects of Kir 1.1a 331X on wild-type Kir 1.1a. Ba2+-sensitive inward current is plotted (I/Io, Vm = −90 mV) as a function of Fmut, the mutant fraction of the total cRNA injected [Fmut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)]. Fmut was adjusted by coinjecting variable amounts of Kir 1.1a 331X with a constant dose of Kir 1.1a (250 pg), and Io is the mean current when Fmut = 0. The dotted line (A) represents the predicted relationship if inclusion of one or more mutant subunits within a tetramer inhibits channel function [I/Io = (1 Fmut)4]. The dashed curve (B) is predicted if two or more subunits are required [I/Io = (1 − Fmut)4 + 4(1 − Fmut)3Fmut]. The data are best fit by an intermediate model, I/Io = (1 − 0.6 Fmut)4, represented by the solid line (C). The coefficient, k, is less than unity, indicating that a single mutant subunit partially inhibits current or that Kir 1.1a331X oligomerizes with a reduced efficiency.
Mentions: To gain further insight into the dominant negative mechanism, increasing doses of mutant cRNA were coinjected with a constant amount of wild-type transcript (Fig. 3; n = 5–14 each dose). Because Kir 1.1a channels are known to be tetrameric in structure (Glowatzki et al. 1995), coinjection should produce five discrete populations, containing zero to four mutant subunits. Assuming random assembly, a binomial probability distribution defined by two parameters, Fmut and n, determines the probability of obtaining each channel population. Fmut, the mutant fraction of the total cRNA injected [Fmut = nanograms mut cRNA/(nanograms mut cRNA + nanograms wt cRNA)], is the probability of incorporating a Kir 1.1a 331X subunit. For a tetrameric channel, the number of subunits, n, is equal to 4. The relative frequency of functional populations determines the resultant macroscopic current density. The predicted relationships for two specific dominant negative models are shown in Fig. 3. Consider the situation when incorporation of one or more mutant subunits abolishes channel activity. The relative current is determined by the probability of assembling four wild-type subunits into a tetramer. In this case, the macroscopic current can be predicted by the relationship, I/Io = (1 − Fmut)4 (Fig. 3, line A). If two or more mutants must be incorporated within a tetramer to inhibit channel activity, an additional term, the probability of forming a channel with one mutant and three wild-type subunits [4 Fmut(1 − Fmut)3], is added to the equation above (line 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