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Evidence for sequential ion-binding loci along the inner pore of the IRK1 inward-rectifier K+ channel.

Shin HG, Xu Y, Lu Z - J. Gen. Physiol. (2005)

Bottom Line: Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K(+) selectivity filter.As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to approximately 10 A.We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site approximately 100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvasnia, 3700 Hamilton Walk, Philadelphia, PA 19104, USA.

ABSTRACT
Steep rectification in IRK1 (Kir2.1) inward-rectifier K(+) channels reflects strong voltage dependence (valence of approximately 5) of channel block by intracellular cationic blockers such as the polyamine spermine. The observed voltage dependence primarily results from displacement, by spermine, of up to five K(+) ions across the narrow K(+) selectivity filter, along which the transmembrane voltage drops steeply. Spermine first binds, with modest voltage dependence, at a shallow site where it encounters the innermost K(+) ion and impedes conduction. From there, spermine can proceed to a deeper site, displacing several more K(+) ions and thereby producing most of the observed voltage dependence. Since in the deeper blocked state the leading amine group of spermine reaches into the cavity region (internal to the selectivity filter) and interacts with residue D172, its trailing end is expected to be near M183. Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K(+) selectivity filter. As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to approximately 10 A. For technical simplicity, we used tetraethylammonium (TEA) as an initial probe to test whether the corresponding residue in IRK1, F254, forms the shallower site. We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site approximately 100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine. These results establish the evidence for physically separate, sequential ion-binding loci along the long inner pore of IRK1, and strongly suggest that the aromatic side chains of F254 underlie the likely innermost binding locus for both blocker and K(+) ions in the cytoplasmic pore.

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Voltage dependence of spermine inhibition of channels with and without a mutation at F254 or D255. The (averaged) fraction of current not blocked by 0.1 mM spermine is plotted against membrane voltage for wild-type and F254A and D255A mutant channels. The curve through the data for wild-type channels is a fit of Eq. 1; those for the mutant channels are fits of a Boltzmann function (with parameters appKd, appZ, and an offset of ∼5% to account for the shallow state with much reduced spermine affinity), yielding K1 = 1.30 (±0.06) × 10−4 M and K2 = 1.44 (±0.13) × 10−2 with valences Z1 = 0.40 ± 0.02 and Z2 = 4.23 ± 0.13 (mean ± SEM; n = 9) for wild type, appKd = 3.82 (±0.49) × 10−6 M and valence appZ = 3.96 ± 0.15 (n = 5) for F254A, and appKd = 2.45 (±0.28) × 10−6 M and appZ = 4.75 ± 0.14 (n = 8) for D255A.
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fig9: Voltage dependence of spermine inhibition of channels with and without a mutation at F254 or D255. The (averaged) fraction of current not blocked by 0.1 mM spermine is plotted against membrane voltage for wild-type and F254A and D255A mutant channels. The curve through the data for wild-type channels is a fit of Eq. 1; those for the mutant channels are fits of a Boltzmann function (with parameters appKd, appZ, and an offset of ∼5% to account for the shallow state with much reduced spermine affinity), yielding K1 = 1.30 (±0.06) × 10−4 M and K2 = 1.44 (±0.13) × 10−2 with valences Z1 = 0.40 ± 0.02 and Z2 = 4.23 ± 0.13 (mean ± SEM; n = 9) for wild type, appKd = 3.82 (±0.49) × 10−6 M and valence appZ = 3.96 ± 0.15 (n = 5) for F254A, and appKd = 2.45 (±0.28) × 10−6 M and appZ = 4.75 ± 0.14 (n = 8) for D255A.

Mentions: We now examine the effects of mutations at F254 and D255 on channel block by intracellular spermine. Fig. 8 shows currents of wild-type and mutant (F254A or D255A) channels in the absence or presence of 0.1 mM spermine. At this concentration, spermine almost completely blocks the outward currents of all three mutant channels. It also slightly inhibits inward currents in wild-type channels but imperceptibly in mutant channels. The normalized currents are plotted against membrane voltage for both wild-type and mutant channels in Fig. 9. As shown previously (Lopatin et al., 1995; Xie et al., 2002; Shin and Lu, 2005), the relation for wild-type channels exhibits both a shallow and a steep phase. However, that shallow phase is apparently eliminated by the mutations. Thus, mutations at both F254 and D255 dramatically disrupt spermine interaction with the shallow locus.


Evidence for sequential ion-binding loci along the inner pore of the IRK1 inward-rectifier K+ channel.

Shin HG, Xu Y, Lu Z - J. Gen. Physiol. (2005)

Voltage dependence of spermine inhibition of channels with and without a mutation at F254 or D255. The (averaged) fraction of current not blocked by 0.1 mM spermine is plotted against membrane voltage for wild-type and F254A and D255A mutant channels. The curve through the data for wild-type channels is a fit of Eq. 1; those for the mutant channels are fits of a Boltzmann function (with parameters appKd, appZ, and an offset of ∼5% to account for the shallow state with much reduced spermine affinity), yielding K1 = 1.30 (±0.06) × 10−4 M and K2 = 1.44 (±0.13) × 10−2 with valences Z1 = 0.40 ± 0.02 and Z2 = 4.23 ± 0.13 (mean ± SEM; n = 9) for wild type, appKd = 3.82 (±0.49) × 10−6 M and valence appZ = 3.96 ± 0.15 (n = 5) for F254A, and appKd = 2.45 (±0.28) × 10−6 M and appZ = 4.75 ± 0.14 (n = 8) for D255A.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2266567&req=5

fig9: Voltage dependence of spermine inhibition of channels with and without a mutation at F254 or D255. The (averaged) fraction of current not blocked by 0.1 mM spermine is plotted against membrane voltage for wild-type and F254A and D255A mutant channels. The curve through the data for wild-type channels is a fit of Eq. 1; those for the mutant channels are fits of a Boltzmann function (with parameters appKd, appZ, and an offset of ∼5% to account for the shallow state with much reduced spermine affinity), yielding K1 = 1.30 (±0.06) × 10−4 M and K2 = 1.44 (±0.13) × 10−2 with valences Z1 = 0.40 ± 0.02 and Z2 = 4.23 ± 0.13 (mean ± SEM; n = 9) for wild type, appKd = 3.82 (±0.49) × 10−6 M and valence appZ = 3.96 ± 0.15 (n = 5) for F254A, and appKd = 2.45 (±0.28) × 10−6 M and appZ = 4.75 ± 0.14 (n = 8) for D255A.
Mentions: We now examine the effects of mutations at F254 and D255 on channel block by intracellular spermine. Fig. 8 shows currents of wild-type and mutant (F254A or D255A) channels in the absence or presence of 0.1 mM spermine. At this concentration, spermine almost completely blocks the outward currents of all three mutant channels. It also slightly inhibits inward currents in wild-type channels but imperceptibly in mutant channels. The normalized currents are plotted against membrane voltage for both wild-type and mutant channels in Fig. 9. As shown previously (Lopatin et al., 1995; Xie et al., 2002; Shin and Lu, 2005), the relation for wild-type channels exhibits both a shallow and a steep phase. However, that shallow phase is apparently eliminated by the mutations. Thus, mutations at both F254 and D255 dramatically disrupt spermine interaction with the shallow locus.

Bottom Line: Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K(+) selectivity filter.As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to approximately 10 A.We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site approximately 100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvasnia, 3700 Hamilton Walk, Philadelphia, PA 19104, USA.

ABSTRACT
Steep rectification in IRK1 (Kir2.1) inward-rectifier K(+) channels reflects strong voltage dependence (valence of approximately 5) of channel block by intracellular cationic blockers such as the polyamine spermine. The observed voltage dependence primarily results from displacement, by spermine, of up to five K(+) ions across the narrow K(+) selectivity filter, along which the transmembrane voltage drops steeply. Spermine first binds, with modest voltage dependence, at a shallow site where it encounters the innermost K(+) ion and impedes conduction. From there, spermine can proceed to a deeper site, displacing several more K(+) ions and thereby producing most of the observed voltage dependence. Since in the deeper blocked state the leading amine group of spermine reaches into the cavity region (internal to the selectivity filter) and interacts with residue D172, its trailing end is expected to be near M183. Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K(+) selectivity filter. As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to approximately 10 A. For technical simplicity, we used tetraethylammonium (TEA) as an initial probe to test whether the corresponding residue in IRK1, F254, forms the shallower site. We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site approximately 100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine. These results establish the evidence for physically separate, sequential ion-binding loci along the long inner pore of IRK1, and strongly suggest that the aromatic side chains of F254 underlie the likely innermost binding locus for both blocker and K(+) ions in the cytoplasmic pore.

Show MeSH
Related in: MedlinePlus