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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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Truncation of amino acids 332–391 (Kir 1.1a 331X) disrupts Kir 1.1a channel functional expression. (A) Representative families of whole-cell currents and (B) macroscopic current-voltage relationships measured from Xenopus oocytes injected with either Kir 1.1a (•) or Kir 1.1a 331X (▪) cRNA (250 pg) using two-microelectrode voltage clamp. Currents were elicited by 500-ms voltage clamp pulses from −150 to +50 mV in 20-mV increments (VHOLD = 0 mV; [K+]o = 45 mM). This pulse protocol was used in all subsequent measurements of macroscopic channel activity, unless otherwise noted.
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Figure 1: Truncation of amino acids 332–391 (Kir 1.1a 331X) disrupts Kir 1.1a channel functional expression. (A) Representative families of whole-cell currents and (B) macroscopic current-voltage relationships measured from Xenopus oocytes injected with either Kir 1.1a (•) or Kir 1.1a 331X (▪) cRNA (250 pg) using two-microelectrode voltage clamp. Currents were elicited by 500-ms voltage clamp pulses from −150 to +50 mV in 20-mV increments (VHOLD = 0 mV; [K+]o = 45 mM). This pulse protocol was used in all subsequent measurements of macroscopic channel activity, unless otherwise noted.

Mentions: As a first approach to examine the functional consequences of COOH-terminal truncation, wild-type Kir 1.1a and Kir 1.1a 331X cRNAs were independently injected into Xenopus oocytes, and macroscopic currents were measured using two-microelectrode voltage clamp. As predicted from the link to Bartter's syndrome, truncation of the extreme COOH terminus of Kir 1.1a abolishes channel activity (Fig. 1). Oocytes injected with Kir 1.1a cRNA-expressed large, weakly inward rectifying macroscopic currents, typical of the wild-type channel (Ho et al. 1993). In contrast, no currents above the background could be detected in oocytes injected with Kir 1.1a 331X cRNA. The mean Ba2+-sensitive macroscopic current at −90 mV was −0.11 ± 0.05 μA (n = 6), compared with −17.95 ± 2.87 μA (n = 12) for the wild-type channel. Consistent with the macroscopic data, no significant activity, except for occasional endogenous stretch-activated channel openings (Yang and Sachs 1990), was detected in oocytes injected with the mutant cRNA (see Fig. 6 C, n = 12).


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)

Truncation of amino acids 332–391 (Kir 1.1a 331X) disrupts Kir 1.1a channel functional expression. (A) Representative families of whole-cell currents and (B) macroscopic current-voltage relationships measured from Xenopus oocytes injected with either Kir 1.1a (•) or Kir 1.1a 331X (▪) cRNA (250 pg) using two-microelectrode voltage clamp. Currents were elicited by 500-ms voltage clamp pulses from −150 to +50 mV in 20-mV increments (VHOLD = 0 mV; [K+]o = 45 mM). This pulse protocol was used in all subsequent measurements of macroscopic channel activity, unless otherwise noted.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Truncation of amino acids 332–391 (Kir 1.1a 331X) disrupts Kir 1.1a channel functional expression. (A) Representative families of whole-cell currents and (B) macroscopic current-voltage relationships measured from Xenopus oocytes injected with either Kir 1.1a (•) or Kir 1.1a 331X (▪) cRNA (250 pg) using two-microelectrode voltage clamp. Currents were elicited by 500-ms voltage clamp pulses from −150 to +50 mV in 20-mV increments (VHOLD = 0 mV; [K+]o = 45 mM). This pulse protocol was used in all subsequent measurements of macroscopic channel activity, unless otherwise noted.
Mentions: As a first approach to examine the functional consequences of COOH-terminal truncation, wild-type Kir 1.1a and Kir 1.1a 331X cRNAs were independently injected into Xenopus oocytes, and macroscopic currents were measured using two-microelectrode voltage clamp. As predicted from the link to Bartter's syndrome, truncation of the extreme COOH terminus of Kir 1.1a abolishes channel activity (Fig. 1). Oocytes injected with Kir 1.1a cRNA-expressed large, weakly inward rectifying macroscopic currents, typical of the wild-type channel (Ho et al. 1993). In contrast, no currents above the background could be detected in oocytes injected with Kir 1.1a 331X cRNA. The mean Ba2+-sensitive macroscopic current at −90 mV was −0.11 ± 0.05 μA (n = 6), compared with −17.95 ± 2.87 μA (n = 12) for the wild-type channel. Consistent with the macroscopic data, no significant activity, except for occasional endogenous stretch-activated channel openings (Yang and Sachs 1990), was detected in oocytes injected with the mutant cRNA (see Fig. 6 C, n = 12).

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