Chang HK , Iwamoto M , Oiki S , Shieh RC .

	Figure 1. Homology model of Kir2.1 channels.(A) Construction of the model was based on a sequence alignment with the structure of a Kir2.2 channel51. Two of the four subunits of the Kir2.1 channel are shown. The channel pore consists of the indicated selectivity filter, central cavity and cytoplasmic pore. Residues involved in polyamine binding are shown in ball-and-chain models and are highlighted by yellow markers.
	Figure 2. Comparison of single-channel conductance in wild-type Kir2.1 channels and E224K/H226E mutants.(a) Current traces and the all-point-histograms of the wild-type and the E224K/H226E mutant at the indicated voltages. At all of the voltages tested, the single-channel current (i, left panels) was smaller in the mutant than in the wild-type channel. However, the probability of the opening of the E224K/H226E channel (right panels) did not seem to be different from that of the wild-type channel. (b) Single-channel current−voltage (i-Vm) relationships at varying K+ concentrations. At low K+ concentrations, the current−voltage curve displays strong inward rectification in the E224K/H226 mutant and mild inward rectification in the wild-type. Symbols (squares, circles and triangles) represent experimental data, and the lines depict the model’s fit to the data. n = 2–6 for both the wild-type and mutant. (c) The [K+]-dependence of the single-channel conductance at −100 mV. The broken lines and solid lines are the fit of the Michaelis-Menten equation and the permeation model (Fig. 6), respectively, to the data with the value at 300 mM as the maximum.
	Figure 3. Measurements of Vstream in the wild-type Kir2.1 channel.(a) The experimental configuration of the coupled water-K+ movement and the resultant negative shift of Vrev when the intracellular osmotic pressure is lower than the extracellular osmotic pressure. (b) Voltage protocol and a set of representative current traces. From a holding potential (hp) of −20 mV, a train of ramp voltage (−20 to +20 mV, rate of change 1 mV/ms) was applied before, during, and after the osmotic pulse. (c) The enlargement of a ramp set (positive and negative ramps) and current traces. Currents recorded under the same intracellular sorbitol concentration (Sori) overlapped with one another. The current−voltage curves obtained from the negative and positive ramps were nearly identical to each other, and the two traces crossed at the same Vrev. (d) Current−voltage relationships (of both ramp branches) at ramp 1, 7 and 13. The curved arrows at the end of the data at +20 mV indicate the transition from the positive to negative ramp. e. The time course of Vrev. Filled and open circles denote data obtained from the positive (●) and negative ramps (○), respectively. The average Vstream values are −1.57± 0.26 mV at 15 mM [K+], with ΔOsm = 1.5 M.
	Figure 4. Measurement of Vstream in the E224K/H226E mutant.(a) Ramp protocol and a set of representative current traces. (b) The enlargement of a ramp set and current traces. Currents recorded under the same intracellular sorbitol concentration (Sori) overlapped one another. (c) Current−voltage relationships (of both ramp branches) at ramps 1, 7 and 13. The curved arrows at the end of the data at +20 mV indicate the transitions from the positive to negative ramp. (d) The time course of Vrev. Filled and open circles denote data obtained from the positive (●) and negative ramps (○).
	Figure 5. Dependence of Vstream on ΔOsm and [K+].(a) The two Vstream – ΔOsm relationships were linear, with slope values of −0.63 ± 0.02 (mean ± standard error) and −0.83 ± 0.07 mV/Osm/kg in the wild-type and the mutant, respectively, at 150 mM [K+]. The line appeared to be steeper in the mutant than in the wild type, although there was no significant difference between the two regression lines (p = 0.07). (b) At 50 mM [K+], the two slope values were significantly different, with −0.82 ± 0.01 in the wild-type and −1.08 ± 0.02 mV/Osm/kg in the mutant (p < 0.05). (c) At 15 mM [K+], the slope value further increased, to −0.98 ± 0.07 and −1.35 ± 0.03 mV/Osm/kg for the wild-type and the mutant, respectively. The two slopes were significantly different (p < 0.05). (d) The relationship between CRw-i and log[K+]. The CRw-i value decreased from 2.15 ± 0.15 at 15 mM [K+] to 1.39 ± 0.04 at 150 mM [K+] (1.80 ± 0.03 at 50 mM [K+]) for the wild-type channel. The CRw-i values for the E224K/H226E mutant changed from 2.98 ± 0.07 at 15 mM [K+] to 1.84 ± 0.15 at 150 mM [K+] (2.37 ± 0.04 at 50 mM [K+]). The black curves were calculated from the model using cycle flux algebra. n = 3 to 6 for wild type; 3 to 4 for E224K/H226E.
	Figure 6. Permeation model, parameters and permeation features for the wild-type and mutant channels.(a) Left panel: The eleven-state permeation diagram for K+ (green circle) and water (red circle) permeation through a Kir2.1 channel (left side, intracellular). Each green channel cartoon represents a K+-water-occupied state in the selectivity filter and cytoplasmic pore. Arrows denote states transitions, which accompany shift movements of K+-water columns. Blue and magenta arrows indicate transitions for the efflux and influx, respectively. Curved lines represent K+ entering from either side to the cavity or the selectivity filter, while curved arrows represent K+ exiting from them. k1 through k10 represent the rate constants for the state transitions. Right panel: Cyclic paths on the permeation diagram. Each cycle was drawn by connecting states, and by completing a cycle, a net transport of K+ and water occurred. In each cycle, the ratio of the coupled movements of water (red number) and K+ (green number) molecules is indicated. For example, cycle a exhibits a coupling ratio of 1:1 and cycle c, 2:1. (b) The affected path and the degree of modification of rate constants in the mutant channel. Solid arrows indicate transition paths with increased rate constants, whereas broken arrows indicate those with decreased rate constants.
	Figure 7. Permeation features for the wild-type and mutant channels.(a) The conductance of the wild-type (left) and mutant (right) channels as a function of the membrane potential at 150 mM K+. The total conductance is nearly flat for the wild-type channel, but it is attenuated at the positive potentials or inward-rectified for the mutant channel. In the wild-type channel, the underlying conductances of the cycle fluxes indicate that the voltage-dependent increasing (cycle c) and decreasing (cycle a and b) cycles compensate for yielding a nearly constant conductance. In the mutant channel, cycle a is attenuated substantially, and the remaining cycles b and c generate attenuated conductance at positive potentials. (b) The relative flux contributions to conductance by various cycles in the wild-type (left) and mutant (right) channels at various [K+].

Ando, Coupled K+-water flux through the HERG potassium channel measured by an osmotic pulse method. 2005, Pubmed

Ando, Coupled K+-water flux through the HERG potassium channel measured by an osmotic pulse method. 2005, Pubmed
Brelidze, A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification. 2003, Pubmed , Xenbase
Chang, The effects of spermine on the accessibility of residues in the M2 segment of Kir2.1 channels expressed in Xenopus oocytes. 2003, Pubmed , Xenbase
Chang, A ring of negative charges in the intracellular vestibule of Kir2.1 channel modulates K+ permeation. 2005, Pubmed , Xenbase
Chang, Charges in the cytoplasmic pore control intrinsic inward rectification and single-channel properties in Kir1.1 and Kir2.1 channels. 2007, Pubmed , Xenbase
Constanti, Fast inward-rectifying current accounts for anomalous rectification in olfactory cortex neurones. 1983, Pubmed
Day, Dendritic excitability of mouse frontal cortex pyramidal neurons is shaped by the interaction among HCN, Kir2, and Kleak channels. 2005, Pubmed
Dhamoon, Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+ current. 2004, Pubmed
Doyle, The structure of the potassium channel: molecular basis of K+ conduction and selectivity. 1998, Pubmed
Fakler, A structural determinant of differential sensitivity of cloned inward rectifier K+ channels to intracellular spermine. 1994, Pubmed , Xenbase
Ficker, Spermine and spermidine as gating molecules for inward rectifier K+ channels. 1994, Pubmed , Xenbase
Fujiwara, Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1. 2006, Pubmed , Xenbase
Guo, Kinetics of inward-rectifier K+ channel block by quaternary alkylammonium ions. dimension and properties of the inner pore. 2001, Pubmed , Xenbase
Hamill, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. 1981, Pubmed
Horie, Voltage-dependent magnesium block of adenosine-triphosphate-sensitive potassium channel in guinea-pig ventricular cells. 1987, Pubmed
Huang, The bundle crossing region is responsible for the inwardly rectifying internal spermine block of the Kir2.1 channel. 2014, Pubmed , Xenbase
Hume, Ionic basis of the different action potential configurations of single guinea-pig atrial and ventricular myocytes. 1985, Pubmed
Iwamoto, Counting ion and water molecules in a streaming file through the open-filter structure of the K channel. 2011, Pubmed
Jogini, Electrostatics of the intracellular vestibule of K+ channels. 2005, Pubmed
John, Mechanism of inward rectification in Kir channels. 2004, Pubmed , Xenbase
Kubo, Control of rectification and permeation by two distinct sites after the second transmembrane region in Kir2.1 K+ channel. 2001, Pubmed , Xenbase
Kubo, Primary structure and functional expression of a mouse inward rectifier potassium channel. 1993, Pubmed , Xenbase
Kuno, Temperature dependence of proton permeation through a voltage-gated proton channel. 2009, Pubmed
Kurata, The polyamine binding site in inward rectifier K+ channels. 2006, Pubmed
Kurata, Locale and chemistry of spermine binding in the archetypal inward rectifier Kir2.1. 2010, Pubmed
Lee, Novel gating mechanism of polyamine block in the strong inward rectifier K channel Kir2.1. 1999, Pubmed , Xenbase
Levitt, Number of water molecules coupled to the transport of sodium, potassium and hydrogen ions via gramicidin, nonactin or valinomycin. 1978, Pubmed
Lopatin, Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. 1994, Pubmed , Xenbase
Lu, Cytoplasmic amino and carboxyl domains form a wide intracellular vestibule in an inwardly rectifying potassium channel. 1999, Pubmed , Xenbase
Matsuda, Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+. , Pubmed
Morais Cabral, Crystal structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain. 1998, Pubmed , Xenbase
Morais-Cabral, Energetic optimization of ion conduction rate by the K+ selectivity filter. 2001, Pubmed
Nimigean, Electrostatic tuning of ion conductance in potassium channels. 2003, Pubmed , Xenbase
Oiki, Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age. 2015, Pubmed
Oiki, A mesoscopic approach to understanding the mechanisms underlying the ion permeation on the discrete-state diagram. 2010, Pubmed
Oiki, Cycle flux algebra for ion and water flux through the KcsA channel single-file pore links microscopic trajectories and macroscopic observables. 2011, Pubmed
Rosenberg, Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes. 1978, Pubmed
Stanfield, A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1. 1994, Pubmed
Tao, Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 A resolution. 2009, Pubmed , Xenbase
Vandenberg, Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions. 1987, Pubmed
Wible, Gating of inwardly rectifying K+ channels localized to a single negatively charged residue. 1994, Pubmed , Xenbase
Yang, Control of rectification and permeation by residues in two distinct domains in an inward rectifier K+ channel. 1995, Pubmed , Xenbase
Yeh, Electrostatics in the cytoplasmic pore produce intrinsic inward rectification in kir2.1 channels. 2005, Pubmed , Xenbase
Zhou, Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. 2001, Pubmed
Zhou, The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates. 2003, Pubmed
Zhou, A mutant KcsA K(+) channel with altered conduction properties and selectivity filter ion distribution. 2004, Pubmed