Xie LH , John SA , Ribalet B , Weiss JN .

R37 HL60025 NHLBI NIH HHS , R37 HL060025 NHLBI NIH HHS

	Figure 1. Stabilization of Kir2.1 channel activity by PIP2. Representative current traces elicited by the ramp pulses between −100 mV and +100 mV from a holding potential of 0 mV are shown. (A) After patch excision into Mg- and polyamine-free bath solution, inward rectification disappeared and current transiently increased before running down. Application of phosphatidylinositol-4,5-bisphosphate (PIP2, 10 μM) after rundown recovered channel activity. (B) Current–voltage (I-V) relationships obtained at times a–d in A. (C) Application of PIP2 (10 μM) immediately after patch excision prevented rundown. (D) MgATP (2 mM), but not K2ATP (2 mM), reactivated current after rundown. (E) After rundown, the PIP2 synthesis inhibitor wortmanin (WMN, 100 μM) prevented reactivation by MgATP (2 mM). The triangle to the right of each trace indicates zero current level. The upward arrow under each trace indicates the patch excision from cell-attached to inside-out configuration. Applications of reagents are indicated by the horizontal bars. The same markings are applicable to subsequent figures.
	Figure 2. Stabilization of Kir2.1 channel activity by long diamines. Ramp pulse–elicited current traces showing that spontaneous rundown after patch excision was prevented by (A) spermine (Spm), (B) diamine 12 (DA12), and (C) diamine 10 (DA10), but not (D) putrescine (Put, DA4), all at 100 μM concentration, holding potential between ramps 0 mV. (E). Normalized current (I/I0) 5 min after excising the patch into control solution (ctl), or solution containing 100 μM Spm, DA12, DA10, Put, or 10 μM PIP2. I0 represents current level at −100 mV before rundown. Numbers of patches are indicated in parentheses above each bar. Data are shown and mean ± SEM. **, P < 0.05 compared with ctl.
	Figure 3. Long polyamines strengthen the PIP2–channel interaction in wild-type Kir2.1 channels. (A) Channel activity shows minimal rundown after reactivated by treatment with 2 mM MgATP. (B) After exposure to 2 mM MgATP to minimize spontaneous rundown, application of PIP2 antibody (PIP2-Ab) induced rundown with a half-time of ∼4 min. (C) DA12 (100 μM) slowed PIP2-Ab–induced rundown. (D) Average time course of rundown induced by PIP2-Ab under control (ctl) and in the presence of DA12 (100 μM). Currents (I) are normalized to the value before PIP2 was added (I0). (E) Ramp pulse–elicited current traces showing differential inhibitory effect of 100 μM neomycin (Neo) in the absence and presence of 100 μM DA10. (F) Neomycin sensitivities in the absence and presence of 100 μM spermine. (G) Neomycin sensitivities in the absence and presence of 100 μM putrescine (Put). (H) Dose–response curves of normalized current (I/I0) versus neomycin concentration in the absence and presence of 100 μM Put, DA10, or spermine. I0 represents current level at −100 mV before perfusion of neomycin. Numbers of patches and mean IC50 are indicated in the legends.
	Figure 4. Long polyamines strengthen the PIP2–channel interaction in Kir2.1/K188Q channels. (A and B) Current traces recorded from the K188Q channels, which have reduced PIP2 binding affinity. Strong channel activation was observed only in the presence of both PIP2 (10 μM) and long diamine (100 μM DA12). The inset in each panel shows the current traces with expanded ordinate scale for clarity. (C) Long polyamines (DA12 and DA10) enhanced channel activity after MgATP treatment. Application of 2 mM MgATP and 100 μM diamines of various lengths are indicated by the horizontal bars above the current traces. (D) Summary of the data shown in the C. The currents are normalized to that in the presence of DA12 (IDA12). **, P < 0.05 compared with ctl. (E) Comparison of time course of neomycin (10 μM) inhibition on Kir2.1 wild type (WT) versus K188Q mutation. Currents were recorded at a holding potential of −80 mV. (F and G) Current traces recorded from K188Q channels showing sensitivities to neomycin under control conditions (ctl, in the absence of any polyamine) and in the presence of 100 μM DA12, respectively. (H) Dose–response curve of normalized current (I/I0) versus neomycin concentration in the absence and presence of 100 μM DA12 in K188Q channels. I0 represents current level at −100 mV before perfusion of neomycin. Number of patches and mean IC50 are indicated in the legends.
	Figure 5. Negative charges at the D172 position, but not E224/E299, are required for the polyamine-induced stabilization of channel open state. (A) Current traces recorded from E224G/E299S mutant channels showing neomycin (Neo) sensitivities in the presence or absence of 100 μM DA10. (B) Normalized current (I/I0)–concentration relationships for neomycin in the E224G/E299S, in the absence (ctl) and presence of 100 μM DA10. Numbers of patches and mean IC50 are indicated in the legends. (C and D) Same as A and B, except for D172N mutant channels.
	Figure 6. Long polyamines do not strengthen the PIP2–channel interaction in Kir1.1 channels. (A) Ramp pulse–elicited current traces were recorded from wild-type Kir1.1 channels. DA10 (100 μM) did not cause any shift in neomycin (Neo) sensitivity. Note the same degree of inhibition by 1 mM neomycin in the absence or presence of 100 μM DA10. (B) Same as A, except for Kir1.1/N171D mutant channels. The extent of inward rectification was enhanced in this mutation. However, DA10 (100 μM) had no effect on neomycin sensitivity. TEA (30 mM) had less effect on the inward currents.
	Figure 7. Schematic model of modulation of Kir channels by PIP2 and long polyamines. Only two M2 helices and two cytoplasmic regions of the tetrameric structure are shown for clarity. The channel opens (O state) when the conserved positively charged residues (e.g., K188, R218) in the cytoplasmic region bind to negatively charged heads of PIP2 molecules, and closes (C) when this interaction is lost. The Kir2.1 has a high open probability of >0.9, suggesting a normally high PIP2 binding affinity. When PIP2 is hydrolyzed by PLC or screened by neomycin (Neo), the channel closes (CN state). The absence of new PIP2 to interact causes channel rundown (R state). Long polyamines (PA) interact with D172 in the M2 region to allosterically strengthen the interaction of the cytoplasmic domain with PIP2, thereby locking the channel in the open configuration (OA state) and preventing rundown or inhibition by neomycin. The channel is blocked at positive membrane potential by polyamine plugging the pore near the selectivity filter (BA state).

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