Yang L et al. (2012), Interactions of external K+ and internal blocke...

XB-ART-46158

J Gen Physiol 2012 Nov 01;1405:529-40. doi: 10.1085/jgp.201210835.

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Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel.

Yang L , Edvinsson J , Palmer LG .

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We investigated the effects of changing extracellular K(+) concentrations on block of the weak inward-rectifier K(+) channel Kir1.1b (ROMK2) by the three intracellular cations Mg(2+), Na(+), and TEA(+). Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K(+) in the inside (cytoplasmic) solution and 11 mM K(+) in the outside (extracellular) solution, these three cations blocked K(+) currents with a range of apparent affinities (K(i) (0) = 1.6 mM for Mg(2+), 160 mM for Na(+), and 1.8 mM for TEA(+)) but with similar voltage dependence (zδ = 0.58 for Mg(2+), 0.71 for Na(+), and 0.61 for TEA(+)) despite having different valences. When external K(+) was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg(2+) but not for Na(+) or TEA(+). In contrast, the N152Y mutation increased the affinity for block by TEA(+) but not for Na(+) or Mg(2+). Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg(2+) but had a small effect on that by Na(+). TEA(+) block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K(+) could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K(+) are explained by shifts in the occupancy of sites within the selectivity filter by K(+) ions.

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Species referenced: Xenopus laevis
Genes referenced: kcnj1 kcnj2

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	Figure 1. Mg2+, Na+, and TEA+ block Kir1.1 channels from the inside with similar voltage dependence. Currents through Kir1.1 channels were measured in inside-out patches with 11 mM K+ plus 99 mM Na+ in the extracellular (pipette) solution and 110 mM K+ in the cytoplasmic (bath) solution, with or without 1 mM Mg2+, 3 mM TEA+, or 20 mM Na+. (A) Typical current traces at voltages of â100 mV and +40 mV. Dashed lines show the closed state of the channel. (B) i-V relationships. Data represent means Â± SEM for 3â10 different patches. (C) Analysis of voltage dependence. Fractional block is linearized and fit to Eq. 3. Linear regression gives estimates of Ki(0) = 1.6 (Mg2+), 1.8 (TEA+), and 160 (Na+) mM, and zÎ´ = 0.58 (Mg2+), 0.61 (TEA+), and 0.72 (Na+).
	Figure 2. Blocking affinity is decreased by external K+. Block was analyzed according to Eq. 3 at extracellular K+ concentrations of 11 and 110 mM for (A) 1 mM Mg2+, (B) 40 mM Na+, and (C) 3 mM TEA+. Typical current traces at two voltages with and without blockers are shown at the top of each panel. Values of Ki(0) increased from 1.6 to 4.2 mM (Mg2+), 117 to 293 mM (Na+), and 1.8 to 5.3 mM (TEA+). No significant changes in zÎ´ were observed for any of the blockers.
	Figure 3. Blocking affinity of Mg2+ is decreased by internal K+. Voltage-dependent reduction in single-channel current was analyzed as in Fig. 1, with 25 or 110 mM K+ in the cytoplasmic (bath) solution. The extracellular (pipette) solution contained 11 mM K+ and zero Na+. (A) Current traces at +40 mV with 25 mM [K+]in with and without 1 mM Mg2+. (B) i-V relationships for the conditions in A. (C) Analysis of voltage dependence. Fractional block is linearized and fit to Eq. 3. Linear regression gives estimates of Ki(0) = 2.38 and zÎ´ = 0.68 (110 mM [K+]in), and Ki(0) = 0.92 and zÎ´ = 0.61 (25 mM [K+]in).
	Figure 4. Adding negative charge to the transmembrane cavity increases affinity for Mg2+ but not for Na+ or TEA+. Block was analyzed in WT and N152D channels according to Eq. 3 at 11 mM of extracellular K+ for (A) 0.2 mM Mg2+, (B) 40 mM Na+, and (C) 3 mM TEA+. Typical current traces at two voltages with and without blockers are shown at the top of each panel. A transition in the presence of TEA+ is magnified in the inset. Values of Ki(0) decreased from 0.81 to 0.11 mM (Mg2+), and increased from 117 to 138 mM (Na+) and 1.8 to 2.2 mM (TEA+). No significant changes in zÎ´ were observed.
	Figure 5. Adding an aromatic residue to the transmembrane cavity increases affinity for TEA+. (A) Typical currents through Kir1.1 N152Y channels in inside-out patches with 110 mM K+ on the outside and 110 mM K+ with and without 0.05 mM TEA+ on the inside of the patch. Currents are shown at â100 and +100 mV. (B) Analysis of Po in the absence and presence of TEA+ (two to five measurements per point). (C) i-V relationships with and without TEA+. (D) Voltage dependence analyzed using Eq. 3 with Po substituted for i for [K+]o = 110 and 11 mM. Values of Ki(0) were 0.37 mM ([K+]o = 110 mM) and 0.11 mM ([K+]o = 11 mM). Values of zÎ´ were 0.82 ([K+]o = 110 mM) and 0.84 ([K+]o = 11 mM).
	Figure 6. Switching the C terminus of Kir1.1 to that of Kir2.1 (C13 chimera) increases the affinity of block by Mg2+ and TEA+. Typical current traces at two voltages with and without blockers are shown at the top of each panel. Block was analyzed according to Eq. 3 at extracellular K+ concentrations of 11 mM for (A) 0.2 mM Mg2+, (B) 40 mM Na+, and (C) 0.01 mM TEA+. Values of Ki(0) decreased from 0.82 to 0.16 mM (Mg2+), 117 to 77 mM (Na+), and 1.8 to 0.05 mM (TEA+). No significant differences in zÎ´ were observed for Na+, or Mg2+, or TEA+ measured from changes in i, but zÎ´ for TEA+ block measured as Po was 1.63.
	Figure 7. Results for block of Kir1.1 by Mg2+ are simulated with a kinetic model for permeation, with blocker binding to the transmembrane cavity. The model is shown in A. In this case, we assume no intrinsic voltage dependence of blocker binding and an electrostatic repulsion of 4.6 RT between ions in the cavity and those in S4. Currents in the presence (red) and absence (blue) of Mg2+ with [K+]o = 1 mM (B), 11 mM (C), and 110 mM (D). Symbols show measured values. Solid lines show predicted currents from the best-fit model parameters. (Inset) Crossover of currents, indicating decreased outward current with increased driving force caused by changes in [K+]o, predicted from the model.
	Figure 8. Results for block of Kir1.1 by Mg2+ and Na+ are simulated with a kinetic model for permeation, with blocker binding to the transmembrane cavity. (A) Fractional block by Mg2+ at different voltages and values of [K+]o. (B) Predicted voltage dependence of Mg2+ block at 110 and 25 mM [K+]in. (C) Currents with [K+]o = 110 mM (red) or 11 mM (blue) and 110 mM of internal K+ with (dashed lines) and without (solid lines) 40 mM Na+. Symbols represent data points. Solid lines show predictions of model parameters listed in Table S2. (D) Predicted occupancies of K+ ions in the S1/S3 positions (red) and S2/S4 positions (blue) in the absence (solid lines) and presence (dashed lines) of 1 mM Mg2+. The black line shows the fraction of unoccupied cavity sites in the absence of Mg2+. The green line indicates the fraction of blocked channels.

References [+] :

Ahern, A cation-pi interaction between extracellular TEA and an aromatic residue in potassium channels. 2006, Pubmed