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Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.

Inanobe A, Nakagawa A, Kurachi Y - PLoS ONE (2013)

Bottom Line: In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants.The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit.Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan ; Center for Advanced Medical Engineering and Informatics, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The cytoplasmic domain of inward rectifier K(+) (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent βD and βH strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K(+) currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit. Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.

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Effects of mutations at position 313 of Kir3.2 on single-channel currents.A. Single-channel recording of the indicated channel at −100 mV. The patch recording in the cell-attached configuration was obtained from HEK293T cells expressing Kir3.2 WT, M313G, and M313T. The holding potential was −100 mV. Open dwell time histograms for each channel are shown at the right side of the trace and are fit with a single exponential function. The single-channel current amplitudes of the WT and M313G mutant are plotted against the membrane potentials.B. Effects of substitutions of threonine 301 in Kir2.1.Met301 in Kir2.1 is equivalent to Met313 in Kir3.2. The substitution of threonine at Met301 in Kir2.1 resulted in a spiky opening with variable conductance. Arrowheads indicate the zero current level.
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pone-0079844-g004: Effects of mutations at position 313 of Kir3.2 on single-channel currents.A. Single-channel recording of the indicated channel at −100 mV. The patch recording in the cell-attached configuration was obtained from HEK293T cells expressing Kir3.2 WT, M313G, and M313T. The holding potential was −100 mV. Open dwell time histograms for each channel are shown at the right side of the trace and are fit with a single exponential function. The single-channel current amplitudes of the WT and M313G mutant are plotted against the membrane potentials.B. Effects of substitutions of threonine 301 in Kir2.1.Met301 in Kir2.1 is equivalent to Met313 in Kir3.2. The substitution of threonine at Met301 in Kir2.1 resulted in a spiky opening with variable conductance. Arrowheads indicate the zero current level.

Mentions: When the expression level of the G protein-gated Kir channels was increased, the cells exhibited the K+ current, even in the absence of receptor stimulation [32,33]. When this basal current amplitude (Ibasal) was divided by that in the presence of 10 µM ACh (Itotal) in every oocyte, the plot of the Ibasal/Itotal fraction against the Itotal showed a linear relationship in the WT and mutant channels when the Itotal was less than 10 µA (Figure 3). Assessment of the basal current fraction of the Kir3.2 WT and mutants only from oocytes with a total current of less than 2.5 μA revealed that many mutants had a higher fraction of Ibasal than the WT (Table 2). This suggests that the presence of Glu236 and Met313 plays a role in stabilizing the closed state, and supports the idea that the conformation of the CPD of Kir3.2 changes around the cytoplasmic pore [10]. However, because the EC50, Hill coefficient and Ibasal/Itotal values of the mutants varied depending on the mutated residues and introduced residues, it is not necessarily appropriate to suggest that the effects of the mutations could simply be compared. Nevertheless, it appears that the Ibasal/Itotal values of some of the Met313 mutants are worthy of note. These mutants contained a small side chain (M313G, M313A, and M313S), exhibited EC50 and Hill coefficient values comparable to those of the WT (Figure 2 and Table 2), and G-V relationship comparable to that of the WT (Figure S2 in File S1). However, reductions in the volume of the side chain dramatically increased the Ibasal/Itotal ratio (Figure 3). To reveal the mechanism underlying this phenomenon, we measured the single-channel activity of the mutants at position 313 in Kir3.2. As shown in Figure 4A, in the cell-attached patch configuration, Kir3.2 WT exhibited a spiky opening with a mean open time of 0.36 ± 0.02 ms (n = 18) and a single-channel conductance of 28 pS [22]. The M313G mutant exhibited a prolonged mean open time of 0.82 ± 0.10 ms (n = 8, p = 0.002) with a single-channel conductance (29 pS), similar to that of the WT. This suggests that the mutation leads to the preferential existence of the open state; moreover, this property could account for the increase in the basal current level. The increased Ibasal may be attributed to endogenous Gβγ trafficking with newly synthesized channels to the plasma membrane [4,32,33]. The mutations in the TMD are also reported to increase the Ibasal [34]. To identify which mechanism, Gβγ-dependent or Gβγ-independent, contributes to the increased Ibasal in M313G, we expressed the Kir3.2 WT and mutant channels in HEK293T cells and measured their activity in the inside-out patch configuration (Figure 5). After excising the patches, ATP was perfused at 2 mM to produce PIP2 at the inner leaflet of the patch membranes. The WT was weakly activated by ATP perfusion and its activity was 15 ± 4% of that evoked by 10 μM GTPγS (n = 7). However, M313G was remarkably stimulated, by 59 ± 6% (n = 5). Taken together, the increase in the affinity to PIP2 is likely to be responsible for the prolonged mean open time and the increased Ibasal of M313G.


Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.

Inanobe A, Nakagawa A, Kurachi Y - PLoS ONE (2013)

Effects of mutations at position 313 of Kir3.2 on single-channel currents.A. Single-channel recording of the indicated channel at −100 mV. The patch recording in the cell-attached configuration was obtained from HEK293T cells expressing Kir3.2 WT, M313G, and M313T. The holding potential was −100 mV. Open dwell time histograms for each channel are shown at the right side of the trace and are fit with a single exponential function. The single-channel current amplitudes of the WT and M313G mutant are plotted against the membrane potentials.B. Effects of substitutions of threonine 301 in Kir2.1.Met301 in Kir2.1 is equivalent to Met313 in Kir3.2. The substitution of threonine at Met301 in Kir2.1 resulted in a spiky opening with variable conductance. Arrowheads indicate the zero current level.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823594&req=5

pone-0079844-g004: Effects of mutations at position 313 of Kir3.2 on single-channel currents.A. Single-channel recording of the indicated channel at −100 mV. The patch recording in the cell-attached configuration was obtained from HEK293T cells expressing Kir3.2 WT, M313G, and M313T. The holding potential was −100 mV. Open dwell time histograms for each channel are shown at the right side of the trace and are fit with a single exponential function. The single-channel current amplitudes of the WT and M313G mutant are plotted against the membrane potentials.B. Effects of substitutions of threonine 301 in Kir2.1.Met301 in Kir2.1 is equivalent to Met313 in Kir3.2. The substitution of threonine at Met301 in Kir2.1 resulted in a spiky opening with variable conductance. Arrowheads indicate the zero current level.
Mentions: When the expression level of the G protein-gated Kir channels was increased, the cells exhibited the K+ current, even in the absence of receptor stimulation [32,33]. When this basal current amplitude (Ibasal) was divided by that in the presence of 10 µM ACh (Itotal) in every oocyte, the plot of the Ibasal/Itotal fraction against the Itotal showed a linear relationship in the WT and mutant channels when the Itotal was less than 10 µA (Figure 3). Assessment of the basal current fraction of the Kir3.2 WT and mutants only from oocytes with a total current of less than 2.5 μA revealed that many mutants had a higher fraction of Ibasal than the WT (Table 2). This suggests that the presence of Glu236 and Met313 plays a role in stabilizing the closed state, and supports the idea that the conformation of the CPD of Kir3.2 changes around the cytoplasmic pore [10]. However, because the EC50, Hill coefficient and Ibasal/Itotal values of the mutants varied depending on the mutated residues and introduced residues, it is not necessarily appropriate to suggest that the effects of the mutations could simply be compared. Nevertheless, it appears that the Ibasal/Itotal values of some of the Met313 mutants are worthy of note. These mutants contained a small side chain (M313G, M313A, and M313S), exhibited EC50 and Hill coefficient values comparable to those of the WT (Figure 2 and Table 2), and G-V relationship comparable to that of the WT (Figure S2 in File S1). However, reductions in the volume of the side chain dramatically increased the Ibasal/Itotal ratio (Figure 3). To reveal the mechanism underlying this phenomenon, we measured the single-channel activity of the mutants at position 313 in Kir3.2. As shown in Figure 4A, in the cell-attached patch configuration, Kir3.2 WT exhibited a spiky opening with a mean open time of 0.36 ± 0.02 ms (n = 18) and a single-channel conductance of 28 pS [22]. The M313G mutant exhibited a prolonged mean open time of 0.82 ± 0.10 ms (n = 8, p = 0.002) with a single-channel conductance (29 pS), similar to that of the WT. This suggests that the mutation leads to the preferential existence of the open state; moreover, this property could account for the increase in the basal current level. The increased Ibasal may be attributed to endogenous Gβγ trafficking with newly synthesized channels to the plasma membrane [4,32,33]. The mutations in the TMD are also reported to increase the Ibasal [34]. To identify which mechanism, Gβγ-dependent or Gβγ-independent, contributes to the increased Ibasal in M313G, we expressed the Kir3.2 WT and mutant channels in HEK293T cells and measured their activity in the inside-out patch configuration (Figure 5). After excising the patches, ATP was perfused at 2 mM to produce PIP2 at the inner leaflet of the patch membranes. The WT was weakly activated by ATP perfusion and its activity was 15 ± 4% of that evoked by 10 μM GTPγS (n = 7). However, M313G was remarkably stimulated, by 59 ± 6% (n = 5). Taken together, the increase in the affinity to PIP2 is likely to be responsible for the prolonged mean open time and the increased Ibasal of M313G.

Bottom Line: In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants.The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit.Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan ; Center for Advanced Medical Engineering and Informatics, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The cytoplasmic domain of inward rectifier K(+) (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent βD and βH strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K(+) currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit. Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.

Show MeSH
Related in: MedlinePlus