Regulation of gating and rundown of HCN hyperpolarization-activated channels by exogenous and endogenous PIP2

Abstract

The voltage dependence of activation of the HCN hyperpolarization-activated cation channels is shifted in inside-out patches by -40 to -60 mV relative to activation in intact cells, a phenomenon referred to as rundown. Less than 20 mV of this hyperpolarizing shift can be due to the influence of the canonical modulator of HCN channels, cAMP. Here we study the role of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in HCN channel rundown, as hydrolysis of PI(4,5)P(2) by lipid phosphatases is thought to underlie rundown of several other channels. We find that bath application of exogenous PI(4,5)P(2) reverses the effect of rundown, producing a large depolarizing shift in HCN2 activation. A synthetic short chain analogue of PI(4,5)P(2), dioctanoyl phosphatidylinositol 4,5-bisphosphate, shifts the HCN2 activation curve to more positive potentials in a dose-dependent manner. Other dioctanoyl phosphatidylinositides with one or more phosphates on the lipid headgroup also shift activation, although phosphatidylinositol (PI) is ineffective. Several lines of evidence suggest that HCN2 is also regulated by endogenous PI(4,5)P(2): (a) blockade of phosphatases slows the hyperpolarizing shift upon patch excision; (b) application of an antibody that binds and depletes membrane PIP(2) causes a further hyperpolarizing shift in activation; (c) the shift in activation upon patch excision can be partially reversed by MgATP; and (d) the effect of MgATP is blocked by wortmannin, an inhibitor of PI kinases. Finally, recordings from rabbit sinoatrial cells demonstrate that diC(8) PI(4,5)P(2) delays the rundown of native HCN currents. Thus, both native and recombinant HCN channels are regulated by PI(4,5)P(2).

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 V_1/2 (ΔV_1/2) 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 V_1/2 of HCN2 depends on number and location of inositol phosphates and on acyl chain length. (A) V_1/2 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) V_1/2 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 V_1/2 and channel kinetics are not independent. (A) Interaction of effects of cAMP and diC₈ PI(4,5)P₂ on V_1/2. Bars show the following (from left to right): cAMP, shift in V_1/2 in response to 10 μM cAMP; cAMP + PIP₂, shift in V_1/2 in response to 10 μM cAMP plus 25 μM diC₈ PI(4,5)P₂; PIP₂ (during cAMP), the difference between the shift in V_1/2 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 V_1/2. V_1/2 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 V_1/2 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 V_1/2 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 V_1/2 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 V_1/2 of +9.5 mV and a t_1/2 of 3.8 min.

Figure 7.

Reducing endogenous PI(4,5)P₂ levels shifts HCN2 activation to more negative voltages. (A) Time course of V_1/2 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 V_1/2 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 V_1/2 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); V_1/2 of HCN2 coexpressed with PI(4)P 5-kinase minus V_1/2 of HCN2 expressed alone (PIP5K); V_1/2 of HCN2 after 30–40-min preincubation in 15 μM wortmannin and 0.05% DMSO minus V_1/2 after preincubation in 0.05% DMSO alone (Wortmannin); V_1/2 after 2-h preincubation in 20 μM LY294002 and 0.1% DMSO minus V_1/2 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, V_1/2 = −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, V_1/2 = −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 V_1/2 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 V_1/2 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, V_1/2 = −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).