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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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Tandem covalent linkage of four wild-type Kir 1.1a subunits constrains channel stoichiometry without altering the biophysical properties. (A) Wild-type Kir 1.1a channels were expressed either as monomers (wt Kir 1.1a) or concatemers (4wt) where four subunits were covalently linked in a head-to-tail fashion via a 10 glutamine residue linker (see text for details). (B) Oocytes injected with cRNA encoding either wt Kir 1.1a (▪) or the 4wt concatemer (□) exhibited weakly inward rectifying macroscopic currents that were blocked in a voltage-dependent manner by Ba2+ (not shown). Shown are currents, normalized to the mean amplitude at −150 mV (I/Io), as a function of voltage. (C) There is no dominant negative effect of Kir 1.1a 331X monomers on concatemeric tetramers, confirming that 4wt channels are comprised of a single tetrameric protein. Shown are currents (Vm = −90 mV) measured in oocytes coinjected with the 4wt concatemer and Kir 1.1a 331X normalized to the mean current in the control group that was coinjected with the 4wt concatemer and an unrelated cRNA, CD4. Representative single channel cell-attached recordings obtained from oocytes injected with either (D) wild-type Kir 1.1a or (E) 4wt concatemer cRNA (top left, Vm = −80 mV). Inward currents are examined so upward deflections represent channel closures. The current–voltage relationship (bottom left), open (top right), and closed (bottom right) dwell-time histograms are shown for each. The single channel conductance of the wild-type and concatemeric channel are identical. The solid line in each dwell-time histogram represents the log-likelihood fit to the experimental data. The kinetics of both channels were best described by single open and closed times, although in some patches a longer lived (τ = 20 ms) closed state, as described by Choe et al. 1998, accounted for <0.5% of channel closures. Both the wild-type and concatemeric channels exhibited a high open probability (0.93 ± 0.01 vs. 0.94 ± 0.004, respectively).
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Figure 4: Tandem covalent linkage of four wild-type Kir 1.1a subunits constrains channel stoichiometry without altering the biophysical properties. (A) Wild-type Kir 1.1a channels were expressed either as monomers (wt Kir 1.1a) or concatemers (4wt) where four subunits were covalently linked in a head-to-tail fashion via a 10 glutamine residue linker (see text for details). (B) Oocytes injected with cRNA encoding either wt Kir 1.1a (▪) or the 4wt concatemer (□) exhibited weakly inward rectifying macroscopic currents that were blocked in a voltage-dependent manner by Ba2+ (not shown). Shown are currents, normalized to the mean amplitude at −150 mV (I/Io), as a function of voltage. (C) There is no dominant negative effect of Kir 1.1a 331X monomers on concatemeric tetramers, confirming that 4wt channels are comprised of a single tetrameric protein. Shown are currents (Vm = −90 mV) measured in oocytes coinjected with the 4wt concatemer and Kir 1.1a 331X normalized to the mean current in the control group that was coinjected with the 4wt concatemer and an unrelated cRNA, CD4. Representative single channel cell-attached recordings obtained from oocytes injected with either (D) wild-type Kir 1.1a or (E) 4wt concatemer cRNA (top left, Vm = −80 mV). Inward currents are examined so upward deflections represent channel closures. The current–voltage relationship (bottom left), open (top right), and closed (bottom right) dwell-time histograms are shown for each. The single channel conductance of the wild-type and concatemeric channel are identical. The solid line in each dwell-time histogram represents the log-likelihood fit to the experimental data. The kinetics of both channels were best described by single open and closed times, although in some patches a longer lived (τ = 20 ms) closed state, as described by Choe et al. 1998, accounted for <0.5% of channel closures. Both the wild-type and concatemeric channels exhibited a high open probability (0.93 ± 0.01 vs. 0.94 ± 0.004, respectively).

Mentions: To critically test the effects of incorporation of a single mutant into a Kir 1.1a channel and determine the origin of the intermediate model, three different tetrameric concatemer cDNAs (4wt, 3wt + 1mut, and 1mut + 3wt) were engineered. By creating tandem concatemers, the functional consequences of incorporating a single mutant subunit within a tetrameric channel can be assessed in a manner that is independent of oligomerization efficiency. As described in methods, four monomeric Kir 1.1a cDNAs were artificially linked together with codons for 10 glutamine residues. Glutamine linkers have been successfully used in previous studies to link potassium channel subunits together (Yang et al. 1995b) and are thought to possess little secondary structure and exert minimal influence on channel function. As shown in Fig. 4, we validated this approach by examining the functional characteristics of 4wt and the effects of Kir 1.1a 331X monomers on concatenated channels.


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)

Tandem covalent linkage of four wild-type Kir 1.1a subunits constrains channel stoichiometry without altering the biophysical properties. (A) Wild-type Kir 1.1a channels were expressed either as monomers (wt Kir 1.1a) or concatemers (4wt) where four subunits were covalently linked in a head-to-tail fashion via a 10 glutamine residue linker (see text for details). (B) Oocytes injected with cRNA encoding either wt Kir 1.1a (▪) or the 4wt concatemer (□) exhibited weakly inward rectifying macroscopic currents that were blocked in a voltage-dependent manner by Ba2+ (not shown). Shown are currents, normalized to the mean amplitude at −150 mV (I/Io), as a function of voltage. (C) There is no dominant negative effect of Kir 1.1a 331X monomers on concatemeric tetramers, confirming that 4wt channels are comprised of a single tetrameric protein. Shown are currents (Vm = −90 mV) measured in oocytes coinjected with the 4wt concatemer and Kir 1.1a 331X normalized to the mean current in the control group that was coinjected with the 4wt concatemer and an unrelated cRNA, CD4. Representative single channel cell-attached recordings obtained from oocytes injected with either (D) wild-type Kir 1.1a or (E) 4wt concatemer cRNA (top left, Vm = −80 mV). Inward currents are examined so upward deflections represent channel closures. The current–voltage relationship (bottom left), open (top right), and closed (bottom right) dwell-time histograms are shown for each. The single channel conductance of the wild-type and concatemeric channel are identical. The solid line in each dwell-time histogram represents the log-likelihood fit to the experimental data. The kinetics of both channels were best described by single open and closed times, although in some patches a longer lived (τ = 20 ms) closed state, as described by Choe et al. 1998, accounted for <0.5% of channel closures. Both the wild-type and concatemeric channels exhibited a high open probability (0.93 ± 0.01 vs. 0.94 ± 0.004, respectively).
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Related In: Results  -  Collection

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

Figure 4: Tandem covalent linkage of four wild-type Kir 1.1a subunits constrains channel stoichiometry without altering the biophysical properties. (A) Wild-type Kir 1.1a channels were expressed either as monomers (wt Kir 1.1a) or concatemers (4wt) where four subunits were covalently linked in a head-to-tail fashion via a 10 glutamine residue linker (see text for details). (B) Oocytes injected with cRNA encoding either wt Kir 1.1a (▪) or the 4wt concatemer (□) exhibited weakly inward rectifying macroscopic currents that were blocked in a voltage-dependent manner by Ba2+ (not shown). Shown are currents, normalized to the mean amplitude at −150 mV (I/Io), as a function of voltage. (C) There is no dominant negative effect of Kir 1.1a 331X monomers on concatemeric tetramers, confirming that 4wt channels are comprised of a single tetrameric protein. Shown are currents (Vm = −90 mV) measured in oocytes coinjected with the 4wt concatemer and Kir 1.1a 331X normalized to the mean current in the control group that was coinjected with the 4wt concatemer and an unrelated cRNA, CD4. Representative single channel cell-attached recordings obtained from oocytes injected with either (D) wild-type Kir 1.1a or (E) 4wt concatemer cRNA (top left, Vm = −80 mV). Inward currents are examined so upward deflections represent channel closures. The current–voltage relationship (bottom left), open (top right), and closed (bottom right) dwell-time histograms are shown for each. The single channel conductance of the wild-type and concatemeric channel are identical. The solid line in each dwell-time histogram represents the log-likelihood fit to the experimental data. The kinetics of both channels were best described by single open and closed times, although in some patches a longer lived (τ = 20 ms) closed state, as described by Choe et al. 1998, accounted for <0.5% of channel closures. Both the wild-type and concatemeric channels exhibited a high open probability (0.93 ± 0.01 vs. 0.94 ± 0.004, respectively).
Mentions: To critically test the effects of incorporation of a single mutant into a Kir 1.1a channel and determine the origin of the intermediate model, three different tetrameric concatemer cDNAs (4wt, 3wt + 1mut, and 1mut + 3wt) were engineered. By creating tandem concatemers, the functional consequences of incorporating a single mutant subunit within a tetrameric channel can be assessed in a manner that is independent of oligomerization efficiency. As described in methods, four monomeric Kir 1.1a cDNAs were artificially linked together with codons for 10 glutamine residues. Glutamine linkers have been successfully used in previous studies to link potassium channel subunits together (Yang et al. 1995b) and are thought to possess little secondary structure and exert minimal influence on channel function. As shown in Fig. 4, we validated this approach by examining the functional characteristics of 4wt and the effects of Kir 1.1a 331X monomers on concatenated channels.

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