Pian P , Bucchi A , Robinson RB , Siegelbaum SA .

HL-28958 NHLBI NIH HHS , NS-36658 NINDS NIH HHS , R01 NS036658 NINDS NIH HHS , P01 HL028958 NHLBI NIH HHS , R56 NS036658 NINDS NIH HHS

	Figure 1. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and dioctanoyl phosphatidylinositol 4,5-bisphosphate (diC8 PI(4,5)P2) cause depolarizing shifts in the activation of HCN2. Macroscopic HCN2 currents are shown for individual inside-out patches in response to a series of hyperpolarizing voltage steps. (A) Before (top) and after (bottom) a 15′ bath application of 1 μM native PI(4,5)P2. (B) Before (top) and after (bottom) a 10′ bath application of 25 μM diC8 PI(4,5)P2. (C and D) Normalized tail currents from A and B, respectively, plotted as a function of test potential and fit with the Boltzmann equation. Data obtained in absence (filled circles) or presence (open circles) of PI(4,5)P2 and diC8 PI(4,5)P2 application. Insets show expanded records of tail currents from A and B, measured at −40 mV.
	Figure 2. Action of diC8 PI(4,5)P2 on the gating of HCN2. (A) Dose–response curve for shift in V1/2 (ΔV1/2) as a function of concentration of diC8 PI(4,5)P2. Data fit with the Hill equation. (B) Time constants (τ) of HCN2 current activation (left) and deactivation (right) in absence (squares, solid line) and presence (circles, dashed line) of 25 μM diC8 PI(4,5)P2. The difference between the time constants of activation at −135 mV before (0.731 ± 0.079 s) and after (1.28 ± 0.22 s) diC8 PI(4,5)P2 application were statistically significant (*, P < 0.02, t test). The difference between the time constants of deactivation at −40 mV before (67.6 ± 5.0 ms) and after (136 ± 13 ms) diC8 PI(4,5)P2 application were also statistically significant (*, P < 0.001, t test).
	Figure 3. Shift in V1/2 of HCN2 depends on number and location of inositol phosphates and on acyl chain length. (A) V1/2 shifts with 10-min applications of dioctanoyl phosphatidylinositol (PI) (−0.3 ± 1.7 mV, n = 3), dioctanoyl phosphatidylinositol 4-phosphate (PI(4)P) (+7.5 ± 2.0 mV, n = 6), dioctanoyl phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) (+12.3 ± 1.8 mV, n = 7), dioctanoyl phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and dioctanoyl phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (+9.3 ± 1.8 mV, n = 8). Differences between the responses to diC8 PI(4,5)P2 and diC8 PI(3,4,5)P3 and between the responses to diC8 PI(4,5)P2 and diC8 PI(4)P are statistically significant (P < 0.05; ANOVA, Post Hoc). (B) V1/2 shifts after 28-min application of 1 μM of native PI(4,5)P2 (+31.8 ± 5.1 mV, n = 4), 10-min application of 25 μM diC8 PI(4,5)P2 (+15.4 ± 0.8 mV, n = 12), and 2-min application of 1 μM oleoyl coA (+16.0 ± 1.7 mV, n = 4).
	Figure 4. Effects of cAMP and diC8 PI(4,5)P2 on V1/2 and channel kinetics are not independent. (A) Interaction of effects of cAMP and diC8 PI(4,5)P2 on V1/2. Bars show the following (from left to right): cAMP, shift in V1/2 in response to 10 μM cAMP; cAMP + PIP2, shift in V1/2 in response to 10 μM cAMP plus 25 μM diC8 PI(4,5)P2; PIP2 (during cAMP), the difference between the shift in V1/2 in response to cAMP plus diC8 PI(4,5)P2 and the shift produced by cAMP alone; PIP2, shift in response to 25 μM diC8 PI(4,5)P2 alone; PIP2 (HCN2/FPN), shift of mutant channel in response to 25 μM diC8 PI(4,5)P2. The shift with diC8 PI(4,5)P2 in the presence of cAMP is less than the shift with diC8 PI(4,5)P2 alone (PIP2) (*, P < 0.01, t test). Error bars show SEM. Number of experiments shown in each bar. (B) Interaction of effects of cAMP and diC8 PI(4,5)P2 on time constants (τ) of HCN2 activation (left axis) and deactivation (right axis). Squares, data obtained in absence of PIP2 and cAMP (solid line, n = 14); circles, data obtained in the presence of 25 μM diC8 PI(4,5)P2 (dashed line, n = 10); triangles, data obtained in presence of 10 μM cAMP (dotted line, n = 4); inverted triangles, data obtained in combined presence of 10 μM cAMP plus 25 μM diC8 PI(4,5)P2 (dash dotted line, n = 4). The difference between the time constant of activation at −105 mV in the presence of cAMP (1.49 ± 0.20 s) versus that in the presence of cAMP and PIP2 (0.664 ± 0.056 s) was statistically significant (*, P < 0.02, t test). The difference between the time constant of activation at −95 mV in the presence of cAMP (5.66 ± 0.48 s) and in the presence of cAMP and PIP2 (2.18 ± 0.33 s) was also statistically significant (**, P < 0.001, t test).
	Figure 5. Phosphatase inhibitors delay the rundown in V1/2. V1/2 values were assessed after inside-out patches were excised into normal bath solution (circles) or FV bath solution to inhibit phosphatase activity (squares). The differences in respective V1/2 values measured 1 and 3 min after patch excision were statistically significant (*, P < 0.05, t test).
	Figure 6. Application of MgATP to inside-out patches shifts HCN2 activation to more positive voltages. (A) Time course of V1/2 of HCN2 in a single inside-out patch experiment showing rundown and response to MgATP. Experiment performed in FV solution to inhibit endogenous phosphatase activity. (B) Time course of average V1/2 shift after bath application of MgATP in absence (squares) or presence (inverted triangles) of 15 μM wortmannin to block PI kinases. For data in response to MgATP alone, the single exponential fit yielded values for the steady-state shift in V1/2 of +9.5 mV and a t1/2 of 3.8 min.
	Figure 7. Reducing endogenous PI(4,5)P2 levels shifts HCN2 activation to more negative voltages. (A) Time course of V1/2 in a single inside-out patch experiment in response to bath application of 30.3 μg/ml of anti-PI(4,5)P2 antibody. (B) Time course of average V1/2 shifts after bath application of anti-PI(4,5)P2 antibody (squares) or heat-inactivated antibody (inverted triangles).
	Figure 8. Summary of effects of altering PI(4,5)P2 levels on HCN2 gating. Average shifts in V1/2 shown in response to various agents using either inside-out patches (gray bars) or whole oocytes (open bars). From top to bottom: bath only (no treatment); 2 mM MgATP (in FV solution, 0.05% DMSO); 2 mM MgATP plus 15 μM wortmannin (in FV solution, 0.05% DMSO); 25 μg/ml poly-d-lysine; 30.3 μg/ml anti-PI(4,5)P2 antibody (αPIP2 Ab); 30.3 μg/ml heat-inactivated anti-PI(4,5)P2 antibody; 30.3 μg/ml anti-granulocyte macrophage colony stimulating factor antibody (αGM-CSF Ab); V1/2 of HCN2 coexpressed with PI(4)P 5-kinase minus V1/2 of HCN2 expressed alone (PIP5K); V1/2 of HCN2 after 30–40-min preincubation in 15 μM wortmannin and 0.05% DMSO minus V1/2 after preincubation in 0.05% DMSO alone (Wortmannin); V1/2 after 2-h preincubation in 20 μM LY294002 and 0.1% DMSO minus V1/2 after preincubation in 0.1% DMSO alone (LY294002). Error bars show SEM. Number of experiments shown next to each bar (n).
	Figure 9. Effect of diC8 PI(4,5)P2 on the rundown of the voltage dependence of activation for HCN currents in sinoatrial node cells. (A) Representative whole-cell currents evoked by stepping the membrane from a holding potential of −35 mV to voltages ranging from −25 to −70 mV in 15-mV increments. The recordings were acquired 1 min (top) and 14 min (bottom) after membrane rupture in the absence (left) and in the presence (right) of 200 μM diC8 PI(4,5)P2 in the pipette solution. Hyperpolarizing test voltages indicated at right. (B) Mean tail current activation curves at 1 min (filled symbols) and 14 min (open symbols) in the absence (squares) and in the presence of 200 μM diC8 PI(4,5)P2 (circles). The curves show best fits of the Boltzmann equation. Mean values of parameters from Boltzmann fits of individual activation curves obtained 1 min after patch rupture in absence and presence of diC8 PI(4,5)P2 were, respectively, V1/2 = −58.0 ± 1.5 mV (n = 8) and −58.3 ± 1.7 mV (n = 8, P > 0.05); s = 8.5 ± 0.7 mV and 7.8 ± 0.2 mV (P > 0.05). Mean parameters from Boltzmann fits obtained after 14 min of whole-cell recording in absence and presence of diC8 PI(4,5)P2 were, respectively, V1/2 = −86.4 ± 2.8 mV (n = 6) and −72.4 ± 2.7 mV (n = 6, P < 0.05); s = 16.8 ± 1.3 mV and 12.9 ± 0.7 mV (P < 0.05). (C) Time course of rundown in V1/2 during whole cell recordings in the absence (squares) and in the presence (circles) of 200 μM diC8 PI(4,5)P2. The two curves differ significantly at times indicated by asterisks. Values of V1/2 for 1- and 14-min points from B. For 8-min points, mean activation parameters in absence and presence of diC8 PI(4,5)P2 were, respectively, V1/2 = −70.9 ± 1.7 mV (n = 8) and −63.4 ± 1.6 mV (n = 8, P < 0.05) and s = 14.1 ± 0.3 mV and 10.9 ± 0.6 mV (P < 0.05). Data for B and C obtained from three animals. There was no difference in mean capacitance between the two groups (not depicted).

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