Yeh SH , Chang HK , Shieh RC .

	Figure 1. Homology model of Kir2.1 structure. The model was constructed based on a sequence alignment with the structure of a KirBac1.1 channel (Kuo et al., 2003). Two of the four subunits of a Kir2.1 channel are shown. Residues involved in spermine and Ba2+ bindings are shown in ball-and-chain models.
	Figure 2. Neutral and positive residues at site 224 induce inward rectification in Kir2.1 channel. (A) Macroscopic currents recorded from inside-out patches containing the wild type, E224G, and E224K mutants in 100 mM symmetrical [K+]. Patches were held at 0 mV, prepulsed to −80 mV for 12 ms and then stepped to Vm ranging from −200 to +100 mV for 20 ms. Capacitive and leak currents were corrected by subtracting the currents recorded after complete channel rundown from those measured during channel activities. (B) I-V relationships of the wild type, E224G, and E224K mutants. Currents were normalized to that at −200 mV. n = 5–7.
	Figure 3. MTSET remains accessible to T141C when spermine inhibits K+ efflux in the T141C/D172N mutant. Current traces in the T141C/D172N mutant exposed to the control (A) and 100 μM spermine (B) solution. Current was recorded at −140 mV from a holding potential of +40 mV before (solid line) and after (dotted line) MTSET treatment (1 mM). The number of seconds on the bottom of each dotted line indicates the time after MTSET treatment. The time courses of MTSET modification in the control and spermine are shown in the right panels. The horizontal lines indicate the zero current levels. (C) In the presence of 100 μM spermine, currents were monitored for 50 s before MTSET treatment. MTSET was applied for 100 s as the patch was held at +40 mV without pulsing. Thereafter, MTSET was washed out and currents were recorded with brief hyperpolarizing pulse to −140 mV. (D) Time courses of MTSET reaction in T141C mutants in control and spermine as indicated.
	Figure 4. Inward rectification with E224K mutation is not due to channel block. (A and C) Macroscopic currents recorded from inside-out patches containing the indicated channel. (B and D) I-V relationship of the indicated channel. n = 3–6.
	Figure 5. Charges at residues 224 control inward rectification of the Kir2.1 channel. Currents through the E224C mutant before and after treatment with MTSET (A) or MTSES (C) and after washout were recorded from the same patch. Traces of the IRK1J channel before and after MTSET (B) or MTSES (D) treatment and after washout. (E and F) The I-V relationships of the indicated channel and treatment. n = 3–5.
	Figure 6. Effects of neutral and positive charges at site 224 on selectivity. I-V relationships recorded from the inside-out patches of the indicated channel exposed to 100 mM internal [Tl+], [K+], and [NH4+]. I-V records were obtained by voltage ramps from −200 to +100 mV over 1.4 s. n = 3–8.
	Figure 7. Effects of E224K mutation on MTSET modification to cysteine mutants external and internal to the bundle crossing. (A) A homology model of Kir2.1 structure showing the positions for cysteine mutations. (B) Current traces recorded at −200 mV (5 ms) from the indicated holding potential (pulse frequency 0.2 Hz) in the absence (solid line) and presence (dotted line) of MTSET from the I176C/E224K mutant. (C) MTSET modification rates for various mutants at the indicated holding potential. Currents were measured at the end of the test pulse. n = 3–6. *, P < 0.05; ***, P < 0.005.
	Figure 8. Effects of [K+]o on the inward rectification induced by spermine in the D172N mutant and the E224K mutant. (A) Relative currents (R) were measured at the indicated [K+]o in 100 mM [K+]i with 100 μM spermine as the inducer of inward rectification. The horizontal line indicates R = 0.5. n = 2–3. (B) I-V and g-V relationships of the E224K mutant in the indicated [K+]o.
	Figure 9. High [K+]i increases conductance but cannot correct inward rectification in E224K/H226E mutant. (A) I vs. (Vm–EK) relationships of the E244K/H226E mutant in the presence of 100 mM external [K+] and various [K+]i. Currents through the E224K/H226E mutant exposed to 20 mM or 300 mM [K+]i were always recorded with currents in 100 mM [K+]i in the same patch. Currents were normalized to that in 100 mM [K+]i at Vm–EK = −150 mV in the same patch and then averaged. (B) Effects of [K+]i on inward rectification. The extent of inward rectification is estimated by the ratio of current at Vm–EK = +50 mV to that at −100 mV. n = 5– 11. **, P < 0.01.
	Figure 10. Schematic model for the effects of charges at site 224 on inward rectification. (A) Negative charges at site 224 guide internal K+ entry to the internal pore and lower local electrostatics, thereby facilitating K+ influx and efflux. (B, left) Positive charges at site 224 fail to guide internal K+ entry, raise local energy barrier, and reduce K+–K+ interaction in the selectivity filter. As a result, K+ efflux is inhibited. (B, right) External K+ entry and K+–K+ interaction are not affected by K+ influx. The high energy barrier at the internal vestibule limits K+ influx. Arrow denotes K+ fluxes. The larger the arrow is the greater the flux is.

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