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Figure 1. Truncation of amino acids 332â391 (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 (â¢) or Kir 1.1a 331X (âª) cRNA (250 pg) using two-microelectrode voltage clamp. Currents were elicited by 500-ms voltage clamp pulses from â150 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 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 = â90 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 proteinâgated Kir channel that exerts a dominant negative effect on Kir 3.1, but does not oligomerize with Kir 1.1a.
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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 = â90 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 â Fmut)4 + 4(1 â Fmut)3Fmut]. The data are best fit by an intermediate model, I/Io = (1 â 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.
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Figure 5. Incorporation of single Kir 1.1a 331X mutant at either the NH2- or COOH-terminal position of a tandem tetramer suppresses channel activity. (A) Representative families of whole-cell currents, (B) composite currentâvoltage relationships, and (C) normalized macroscopic Ba2+-sensitive currents (Vm = â90 mV) measured from oocytes injected with equivalent amounts of 4wt, 3wt + 1mut, or 1mut + 3wt concatenated tetramer cRNA (1 ng; *P < 0.0001) are shown.
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Figure 7. Fusion of EGFP to the Kir 1.1a NH2 terminus does not alter channel function. Shown are representative single channel recording (Vm = â80 mV) and corresponding currentâvoltage 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.
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Figure 9. A series of truncated mutants delimit amino acids 332â351 as the COOH-terminal domain necessary to maintain channel function. (A) Kir 1.1a channels were reconstructed by adding residues back to the Bartter's mutant, Kir 1.1a 331X. Amino acids 332â361 represent the most conserved domain (30% identity) within the extreme COOH terminus of inward rectifying K+ channels. (B) Shown are normalized Ba2+-sensitive whole cell currents (Vm = â90 mV) obtained from oocytes expressing each of the truncated mutants depicted in A (*P < 0.01). Replacement of amino acids 332â351 (Kir 1.1a 351X) restored channel activity, while deletion of this domain (Kir 1.1a Î332-351) abolished macroscopic currents.
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Figure 8. COOH-terminal truncation does not alter Kir 1.1a trafficking or plasma membrane stability in the Xenopus oocyte expression system. (A) Representative optical sections acquired from oocytes at a focal plane near the equator using laser-scanning confocal microscopy. Oocytes injected with EGFP, EGFP-Kir 1.1a, or EGFP-Kir 1.1a 331X cRNA were compared with uninjected oocytes. (B) Circumferential fluorescence intensity plotted as a function of the fusion protein cRNA dose injected. No significant differences in circumferential fluorescence distribution and intensity were detected at any injection dose. The solid line represents the linear regression fit of the average circumferential fluorescence for oocytes injected with either wild-type or mutant fusion protein cRNA. C) Macroscopic conductance was measured in oocytes injected with either fusion construct at several cRNA injection doses and is plotted as a function of plasma membrane delimited fluorescence. Slope conductance was determined between â110 and â30 mV from oocytes bathed in 5 mM [K+]o. No conductance above background was detected in oocytes expressing EGFP-Kir 1.1a 331X, consistent with the premise that Kir 1.1a 331X resides in the plasma membrane but is in a nonconductive or inactive conformation.
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Figure 10. COOH-terminal truncation stabilizes an aberrant inactive gating mode. (A) Diary plots of single channel activity in Kir 1.1a 351X vs. the wild type. Shown are plots of open probability measured in 15-s intervals over 20 min. Single channel recordings were obtained in the cell-attached mode (Vm = â80 mV) from oocytes injected with either wild-type or Kir 1.1a 351X cRNA. The mutant exhibits an inactive mode that has a mean lifetime of 5.12 min. (B) Average open probabilities for both wild-type and truncated mutants (n = 4â5) measured over the duration of a 15â20-min recording (*P < 0.0001). The reduced Po of Kir 1.1a 351X results from the stabilization of the long-lived inactive state.
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Figure 11. Upon spontaneous recovery from inactivation, Kir 1.1a 351X channels exhibit wild-type biophysical properties. (A) A typical single channel record (cell attached mode, Vm = â80 mV) illustrating the transition from active to inactive gating conformations. (B) The single channel conductance (left) and mean open (bottom right) and closed (top right) lifetime histograms of Kir 1.1a 351X in the active gating mode are indistinguishable from the wild-type channel (compare with Fig. 4).
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Figure 6. The complete dominant negative effect of one Kir 1.1a 331X is confirmed in single channel recordings. Single channel records and corresponding all-points amplitude histograms were obtained in cell-attached mode at Vm = â80 mV from oocytes injected with either (A) wt Kir 1.1a, (B) 4wt concatenated tetramer, (C) Kir 1.1a 331X, or (D) 3wt + 1mut concatenated tetramer. Occasional small-conductance channel events could be detected in some recordings from oocytes injected with the mutant or mutant-containing concatemer cRNA. These do not likely reflect mutant channel function, as similar events could be detected in uninjected oocytes. Identical results were obtained for both mutant-containing concatemers (1mut + 3wt, not shown).
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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).
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