Li J et al. (2014), Lack of negatively charged residues at the exte...

XB-ART-49539

PLoS One 2014 Jan 01;910:e111372. doi: 10.1371/journal.pone.0111372.

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Lack of negatively charged residues at the external mouth of Kir2.2 channels enable the voltage-dependent block by external Mg2+.

Li J , Xie X , Liu J , Yu H , Zhang S , Zhan Y , Zhang H , Logothetis DE , An H .

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Kir channels display voltage-dependent block by cytosolic cations such as Mg2+ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K+ concentration. In contrast, extracellular Mg2+ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg2+ and the relationship between the Mg2+ blockade and activity potentiation by permeant K+ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg2+ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg2+ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg2+ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg2+ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K+ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg2+.

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Species referenced: Xenopus laevis
Genes referenced: gnl3 kcnj12 kcnj2

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	Figure 2. The kinetics of inhibition and recovery induced by Mg2+ wash-in or wash-out.(A) and (B) are the protocols used to inhibit, activate the currents, respectively. The protocols will repeat until the currents reach the plateau. (C) and (D) are the representative current traces of inhibition and recovery processes. Î and * show the currents of initial and final traces. (E) and (F) are the inhibition and recovery kinetics. Î and * show current corresponding to C and D. (G) shows the summary data of the time constants corresponding to E and F. Data represent means Â± SEM for 7 experiments.
	Figure 3. Mg2+ ion binds at the S0 site of Kir2.2 both in high and physiological conditions.Distributions of extracellular K+ and Mg2+ ions of the initial (A and D) and final (B and E) snapshots. Mg2+ and K+ ions are represented by yellow and red spheres, respectively. In panel B and E, the blue spheres represent the binding Mg2+ ions. (C and F) Evolution of the distance (between the center of the selective filter and each Mg2+) against time (5â25 ns). The total simulation time was 25 ns. In the first 5 ns, the ions are fixed for equilibration of the lipids. The No. 3 Mg 2+ is corresponding to the bound Mg2+.
	Figure 4. K+ ions compete with Mg2+ to bind at the S0 site.Distributions of extracellular K+ and Mg2+ ions of the initial (A) and final (B) snapshots. Mg2+ and K+ ions are represented by yellow and red spheres, respectively. (C) Evolution of the distance (between the center of the selective filter and Mg2+) against time (5â25 ns). The total simulation time was 25 ns. In the first 5 ns, the ions are fixed for equilibration of the lipids.
	Figure 5. There is only one negative ring in Kir2.2 but two in Kir2.1.Sequence alignments for Kir2.1 and Kir2.2 (A) and the schematic positions of the negative rings (B). There are two negative rings in Kir2.1 formed by three negatively charged and one polar amino acid side chains which are E125 & N127 (highlighted in red circle), D152 & E153 (highlighted in blue circle). Whereas, Kir2.2 channel lacks one of the two negatively charged rings (red dotted line circle, bottom right). For clarity, only two of four subunits are shown. (C) Ribbon model of the extracellular side of two subunits of Kir2.2 showing the positions of G119 (purple), Q126 (red) and H128 (red).
	Figure 6. The mutants of Kir2.2 weaken the external Mg2+ blockage.(A) The protocol used for recording the degree of inhibition which was hyperpolarization from 0 mV holding potential to â70 mV for 500 ms, and then stepped to 0 mV. (B) The representative currents trace in Kir2.2 channel. (C) degree of Inhibition (Î³â=â(I0âIf)/I0) for wild type (WT), WT+EGTA, G119D, Q126E, H128D and Triplet mutant (G119D, Q126E and H128D) channels.
	Figure 7. Extracellular K+ density distribution in Kir2.2 and its mutants.The simulations of the Kir2.2 and its mutants were carried out in 0.15 M K+. The extracellular K+ density plots are shown as functions of radial distance from the pore axis (Râ=â(x2+y2)1/2) and height in the simulation box (Z). K+ density distribution in Kir2.2 WT (A), G119D (B), Q126E (C), H128D (D) and Triplet mutant (G119D, Q126E and H128D) channels (E).
	Figure 1. External Mg2+ voltage-dependently blocks the currents through Kir2.2 but not Kir2.1.Whole-cell currents were recorded by two-electrode voltage clamp. (A) Protocol used for activating the currents. The protocol contains a series of 2s-long sweeps. Each sweep consisted of a 50 ms test pulse to â70 mV and 1950 ms conditional pulse from â70 mV to +70 mV in 10 mV steps. There are 15 sweeps for each experiment. The first 50-ms test pulse (â70 mV) is going before the conditional pulses. As a control, the test pulses follow the conditional pulses. (B) and (C) are the representative currents Kir2.2 recorded with and without Mg2+. (D) is the relationship between the currents of Kir2.2 recorded at â70 mV and the corresponding conditional voltage. The data recorded with 1 mM Mg2+ and 0 Mg2+ are shown as black and red lines, respectively. (E) is the representative currents Kir2.1 recorded with Mg2+. (F) is the relationship between the currents of Kir2.1 recorded at â70 mV and the corresponding conditional voltage. Data represent means Â± SEM for 7 experiments.

References [+] :

Apell, Formation of ion channels by a negatively charged analog of gramicidin A. 1977, Pubmed