Shin HG et al. (2005), Evidence for sequential ion-binding loci along ...

XB-ART-1589

J Gen Physiol 2005 Aug 01;1262:123-35. doi: 10.1085/jgp.200509296.

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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 .

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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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???displayArticle.link??? J Gen Physiol
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Species referenced: Xenopus laevis
Genes referenced: eno3 kcnj12 kcnj2 kcnj3

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	Figure 1. Inhibition of IRK1 currents by TEA. Current traces were recorded from a single patch in the absence (control) or presence of TEA at the concentrations indicated. Currents were elicited by stepping membrane voltage from the 0 mV holding potential to â100 mV and then to test voltages between â100 mV and +100 mV in 10-mV increments before returning it to the holding potential. Dotted line indicates the zero current level.
	Figure 2. Voltage dependence of steady-state IRK1 block by TEA. (A) Averaged I-V curves (mean Â± SEM; n = 6) determined at the end of test pulses in the absence (control) or presence of various concentrations of TEA. (B) The fraction of current not blocked is plotted against TEA concentration at the four representative voltages indicated. Curves through the data represent the equation I/Io = appKd/(appKd + [TEA]). (C) The natural logarithm of appKd is plotted against membrane voltage. The curve through the data is a fit of Eq. 1a, yielding K1 = 2.21 (Â±0.18) Ã 10â4 M and K2 = 4.65 Â± 1.41 with valences Z1 = 0.47 Â± 0.03 and Z2 = 1.61 Â± 0.08 (mean Â± SEM; n = 6). The dashed lines indicate the limiting slopes.
	Figure 3. Crystal structure of cytoplasmic termini of GIRK1 (Nishida and MacKinnon, 2002). Shown on the left are two subunits of the cytoplasmic pore whose external end is on top. Side chains are highlighted for residues selenomethionine (Mse)308 (top, yellow), Ser225/Glu300 (middle, red), and Phe255 (bottom, pink), corresponding to Met307, Glu224/Glu299, and Phe254 in IRK1. Shown on the right are the center regions of cross sections through the tetrameric structure at the three levels indicated.
	Figure 4. Effects of E224G or E299S mutations on voltage-dependent channel block by TEA. (A) Current traces for each type of mutant channel were recorded from a single patch in the absence (control) or presence of TEA at the concentrations indicated. Dotted lines indicate the zero current level. (B) The natural logarithm of appKd for the mutant channels (determined as shown in Fig. 2 B) is plotted against membrane voltage. The data for wild-type channels (taken from Fig. 2 C) are plotted here for comparison. The lines through the data for mutant channels are fits of a Boltzmann function. The fits yield appKd = 2.47 (Â±0.57) Ã 10â4 M and valence appZ = 0.63 Â± 0.01 (mean Â± SEM; n = 5) for E224G, and appKd = 2.51 (Â±0.56) Ã 10â4 M and valence appZ = 0.60 Â± 0.04 for E299S (n = 5).
	Figure 5. Inhibition by TEA of currents through channels with a mutation at F254 or D255. Current traces for each type of mutant channel were recorded from a single patch in the absence (control) or presence of TEA at the concentrations indicated. Dotted lines indicate the zero current level.
	Figure 6. Voltage dependence of the apparent TEA affinity of channels with a mutation at F254 or D255. The natural logarithm of appKd for the mutant channels (determined as shown in Fig. 2 B) is plotted against membrane voltage. The data for wild-type channels (taken from Fig. 2 C) are plotted for comparison. The curves through the data for the F254Y and D255A mutant channels are fits of Eq. 1a, whereas lines through the data for F254A and F254V mutant channels are fits of a Boltzmann function (with parameters appKd and appZ). The fits yield K1 = 4.38 (Â±0.39) Ã 10â4 M and K2 = 2.05 Â± 0.82 with valences Z1 = 0.43 Â± 0.06 and Z2 = 1.64 Â± 0.08 (n = 8) for F254Y; K1 = 2.55 (Â±0.25) Ã 10â4 M and K2 = 2.27 Â± 1.07 with Z1 = 0.36 Â± 0.07 and Z2 = 1.27 Â± 0.08 (n = 3) for D255A; appKd = 2.64 (Â±0.20) Ã 10â3 M and valence appZ = 1.42 Â± 0.03 (n = 5) for F254A; appKd = 2.54 (Â±0.41) Ã 10â3 M and appZ = 1.95 Â± 0.07 (n = 6) for F254V.
	Figure 7. Inhibition of F254A mutant channels by a high concentration of TEA. (A) Current traces were recorded from a single patch in the absence (control) or presence of 30 mM TEA. Currents were elicited by stepping membrane voltage from the 0 mV holding potential to â200 mV and then to test voltages between â200 mV and +100 mV in 5-mV increments before returning it to the holding potential. Dotted line indicates the zero current level. (B) The (averaged) fraction of current not blocked by 30 mM TEA is plotted against membrane voltage between â200 and 100 mV. The curve though the data is a fit of a Boltzmann function (with an offset to account for the shallow state with much reduced spermine affinity), yielding appKd = 3.41 (Â±0.19) Ã 10â3 M and valence appZ = 1.34 Â± 0.03 (mean Â± SEM; n = 3).
	Figure 8. Inhibition by spermine of currents through channels with a mutation at F254 or D255. Current traces for wild-type and F254A and D255A mutant channels, each type recorded from a single patch in the absence (control) or presence of 0.1 mM spermine. During recordings, currents were elicited by stepping membrane voltage from the 0 mV holding potential to â100 mV and then to test voltages between â100 and +100 mV in 10-mV increments before returning it to the holding potential. Dotted lines indicate the zero current level.
	Figure 9. 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 â¼5% to account for the shallow state with much reduced spermine affinity), yielding K1 = 1.30 (Â±0.06) Ã 10â4 M and K2 = 1.44 (Â±0.13) Ã 10â2 with valences Z1 = 0.40 Â± 0.02 and Z2 = 4.23 Â± 0.13 (mean Â± SEM; n = 9) for wild type, appKd = 3.82 (Â±0.49) Ã 10â6 M and valence appZ = 3.96 Â± 0.15 (n = 5) for F254A, and appKd = 2.45 (Â±0.28) Ã 10â6 M and appZ = 4.75 Â± 0.14 (n = 8) for D255A.
	Figure 10. Inhibition of A306G-M307G double mutant channels by spermine. (A) Current traces recorded from a single patch in the absence (control) or presence of 0.1 mM spermine. (B) The (averaged) fraction of currents not blocked is plotted against membrane voltage for wild-type (taken from Fig. 9) and mutant channels. The curve through the mutant data is a fit of Eq. 1a, yielding K1 = 9.06 (Â±0.49) Ã 10â5 M and K2 = 1.45 (Â±0.16) Ã 10â2 with valences Z1 = 0.53 Â± 0.02 and Z2 = 4.20 Â± 0.16 (mean Â± SEM; n = 5).
	Figure 11. Inhibition of M183A mutant channels by spermine. (A) Current traces recorded from a single patch in the absence (control) or presence of 0.1 mM spermine. (B) The (averaged) fraction of currents not blocked is plotted against membrane voltage for wild-type (taken from Fig. 9) and mutant channels. The curve though the mutant data is a fit of Eq. 1a, yielding K1 = 2.11 (Â±0.39) Ã 10â4 M and K2 = 9.31 (Â±1.84) Ã 10â2 with valences Z1 = 0.21 Â± 0.06 and Z2 = 2.20 Â± 0.14 (mean Â± SEM; n = 6).
	Figure 12. Models for TEA and spermine block of the IRK1 channel. Binding of a TEA (A) or spermine (SPM; B) molecule to a channel (Ch) produces two sequential blocked states (ChTEA1 and ChTEA2 or ChSPM1 and ChSPM2). K1 = [Ch][X]/[ChX1] and K2 = [ChX1]/[ChX2]. Except for the innermost one, K+ ions are positioned arbitrarily in the inner pore of the accompanying diagrams.
	Figure 13. Predicted blocking properties of mutant channels lacking the shallow or the deep TEA-binding site. The identical solid curves represent a fit of Eq. 1a to the plot of lnappK versus Vm for wild-type channels, taken from Fig. 2 C. The dotted line in A is a simulation of the model with only the shallow site using the first term of Eq. 1a (K1 = 2.21 Ã 10â4 M and Z1 = 0.47), whereas the dotted line in B is a simulation of the model with only the deeper site using the second term of Eq. 1a (K1K2 = 1.02 Ã 10â3 M, Z1 = 0, and Z2 = 1.61). All values of the parameters used in the simulations were taken from Fig. 2 C, except Z1 in B, which was set as zero. The dashed line in B is the limiting slope of the steep phase.
	Figure 14. Comparison of a partial sequence among various Kir channels. Shown is partial sequence alignment among four different Kir channels. Bolded residues correspond to IRK1's F254 and D255.

References [+] :

Choi, The internal quaternary ammonium receptor site of Shaker potassium channels. 1993, Pubmed, Xenbase