Regulation of Gating and Rundown of HCN Hyperpolarization-activated Channels by Exogenous and Endogenous PIP2

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)P2) in HCN channel rundown, as hydrolysis of PI(4,5)P2 by lipid phosphatases is thought to underlie rundown of several other channels. We find that bath application of exogenous PI(4,5)P2 reverses the effect of rundown, producing a large depolarizing shift in HCN2 activation. A synthetic short chain analogue of PI(4,5)P2, 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)P2: (a) blockade of phosphatases slows the hyperpolarizing shift upon patch excision; (b) application of an antibody that binds and depletes membrane PIP2 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 diC8 PI(4,5)P2 delays the rundown of native HCN currents. Thus, both native and recombinant HCN channels are regulated by PI(4,5)P2.

The voltage dependence of activation of the HCN hyperpolarization-activated cation channels is shifted in insideout 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 infl uence 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 fi nd 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 .

I N T R O D U C T I O N
The HCN channel gene family (HCN1-4) encodes the hyperpolarization-activated cation channels that generate the excitatory pacemaker current (I h or I f ), which contributes to the rhythmic activity of both neurons and cardiac myocytes (Robinson and Siegelbaum, 2003;DiFrancesco, 2006). Cyclic AMP has long been known to directly enhance I h by shifting its activation to more positive potentials (DiFrancesco and Tortora, 1991). More recently, HCN channel function has been found to be regulated by several other mechanisms, including tyrosine phosphorylation (Yu et al., 2004;Zong et al., 2005;Arinsburg et al., 2006); association with MiRP1, a potential HCN channel β subunit (Yu et al., 2001); and association with TRIP8b, a cytoplasmic protein that controls channel traffi cking (Santoro et al., 2004). Despite the discovery of these new modes of I h regulation, several lines of evidence, including the phenomenon of channel rundown, suggest the existence of other regulatory mechanisms that have yet to be identifi ed.
The activation of I h shifts by as much as −40 to −60 mV relative to that in intact cells during recordings from inside-out patches or during prolonged dialysis associated with whole-cell recordings, for both native HCN channels in cardiac myocytes (DiFrancesco et al., 1986;DiFrancesco and Tortora, 1991;DiFrancesco and Mangoni, 1994;Bois et al., 1997) and recombinant channels in heterologous cells (Chen et al., 2001). In contrast, this rundown phenomenon is not observed in perforated-patch whole-cell recordings, suggesting that rundown may be due to the loss of intracellular constituents that regulate I h in intact cells (Zhou and Lipsius, 1993). Since cAMP produces, at most, a 20-mV positive shift in the activation of I h , loss of a modulatory infl uence of cyclic nucleotides after patch excision can account for no more than half of the 40-60-mV shift seen during rundown (DiFrancesco and Tortora, 1991;Chen et al., 2001), suggesting the presence of other modulatory factors. Neither MiRP1 (Yu et al., 2001) nor TRIP8b (Santoro et al., 2004) are likely candidates for this factor since these proteins do not act to shift HCN activation gating to more positive potentials in intact cells. Although overexpression of Src tyrosine kinase in HEK-293 cells does shift the voltage dependence of HCN channel activation to more positive Correspondence to Steven A. Siegelbaum: sas8@columbia.edu The online version of this article contains supplemental material.
Abbreviations used in this paper: HCN, hyperpolarization-activated, cyclic nucleotide-regulated, cation-nonselective; PI(4,5)P 2 or PIP 2 , phosphatidylinositol 4,5-bisphosphate; diC 8 , dioctanoyl; PI, phosphatidylinositol; LC acyl-coA, long chain acyl-coenzyme A; K ATP , ATPsensitive K + ; K ir , inwardly rectifying K + . potentials (Arinsburg et al., 2006), loss of Src activity is also unlikely to underlie rundown because pharmacological inhibition of endogenous Src has relatively little effect on the voltage dependence of HCN channel activation in intact cells (Zong et al., 2005;Arinsburg et al., 2006). Thus, the identity of the regulatory mechanism responsible for the rundown of I h activation remains unclear. Interestingly, rundown may be of physiological relevance since a −40-mV hyperpolarizing shift in HCN channel activation is observed in intact ventricular myocytes during postnatal development (Robinson et al., 1997) and this shift does not appear to be due to changes in basal cAMP concentration (Qu et al., 2001). Moreover, during heart failure, HCN channel activation in ventricular myocytes shifts back toward more positive potentials, producing a proarrhythmic increase in pacemaker current (Cerbai et al., 1997).
One promising candidate molecule for underlying rundown is the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Although PI(4,5)P 2 was initially discovered as a precursor for two other second messengers, inositol trisphosphate and diacylglycerol, it has more recently been shown to play an active role in cellular physiology, including enzyme activation, membrane traffi cking, and, more specifi cally, the regulation of ion channels (Hilgemann et al., 2001;McLaughlin et al., 2002;Suh and Hille, 2005). Here we examine the hypothesis that the hyperpolarizing shift in HCN channel gating upon patch excision results from the loss of a basal regulatory action of PI(4,5)P 2 during rundown due to lipid dephosphorylation by membranebound phosphatases. Such a mechanism has been previously found to underlie the functional changes observed upon patch excision for a variety of other ion channels, including inward rectifying K + channels  and P/Q-type voltage-gated Ca 2+ channels (Wu et al., 2002;Suh and Hille, 2005).
To address this hypothesis, we examined both recombinant HCN2 channels expressed in Xenopus oocytes and native HCN currents in cardiac sinoatrial cells. We employed several techniques to manipulate membrane levels of PI(4,5)P 2 in both excised patches and whole oocytes, while monitoring the consequences on HCN2 gating. The results from these experiments are consistent with the idea that HCN2 and native sinoatrial HCN currents are regulated by both exogenous and endogenous PI(4,5)P 2 .

Xenopus Oocyte Electrophysiological Recordings
Cell-free inside-out patches were obtained 5-6 d after cRNA injection, and data were acquired using a patch-clamp amplifi er (Axopatch 200B; Axon Instruments, Inc.). Patch pipettes had resistances of 1-3 MΩ and were fi lled with a pipette solution containing (in mM) 96 KCl, 1 NaCl, 10 HEPES, 1.8 CaCl 2 , and 1 MgCl 2 , pH 7.4. The bath solution contained 96 KCl, 1 NaCl, 10 HEPES, and 5 EGTA, pH 7.4. The FV bath solution used to inhibit phosphatases contained 96 KCl, 0.4 NaCl, 10 HEPES, 5 EGTA, 0.1 KF, 0.2 Na 3 VO 4 , pH 7.4. An Ag-AgCl ground wire was connected to the bath solution by a 3 M KCl agar bridge electrode, and junction potential was compensated before the formation of each patch. Linear leak current was not subtracted. Data were fi ltered at 1 kHz with an 8-pole low-pass Bessel fi lter (Frequency Devices) and sampled at 2 kHz with an ITC-16 interface (Instrutech) and Pulse software (HEKA).
Two microelectrode voltage-clamp recordings were obtained 1 d after cRNA injection using an oocyte clamp amplifi er (model OC-725C; Warner Instruments). Data were fi ltered at 1 kHz and sampled at 2 kHz. The recordings were obtained with the oocytes bathed in an extracellular solution containing (in mM) 96 KCl, 2 NaCl, 10 HEPES, and 2 MgCl 2 , pH 7.5. Microelectrodes were fi lled with 3 M KCl and had resistances of 0.5-2 MΩ.
Hyperpolarizing voltages in 10-mV step increments were applied to either inside-out patches or intact oocytes from a holding potential of −30 mV. All recordings were obtained at room temperature (18-22°C).
Data Analysis HCN currents were analyzed as previously described (Chen et al., 2001). Activation curves were determined from plots of tail current amplitude (measured at −40 mV) as a function of test voltage during 3-s hyperpolarizing steps. Activation curves were fi t with a Boltzmann equation to obtain the midpoint voltage of activation (V 1/2 ) and slope factor (s) of the relation (both in units of mV). Dose-response curves plotting the magnitude of the shift in V 1/2 as a function of diC 8 PI(4,5)P 2 concentration were measured and fi tted with the Hill equation to obtain the maximal shift in V 1/2 at saturating lipid concentration, the concentration of lipid producing a half-maximal shift (K 1/2 ), and the Hill coeffi cient (h). Analysis was done using PulseFit (HEKA), Excel (Microsoft), and Origin (Microcal). Single comparisons between two experimental conditions were evaluated by a Student's t test. Comparisons involving multiple populations were evaluated by ANOVA with a Scheffe Post-Hoc test.

Experiments on Rabbit Sinoatrial Cells
Animal protocols conformed to the guidelines for the care and use of laboratory animals (National Institutes of Health publication no. 85-23). Rabbit sinoatrial cells were isolated as reported (DiFrancesco et al., 1986). Whole-cell patch-clamp experiments were performed using pipettes with a resistance of 2.5-4 MΩ fi lled with a solution containing (in mM) 130 aspartic acid, 146 KOH, 10 NaCl, 2 CaCl 2 , 5 EGTA-KOH, 2 Mg-ATP, and 10 HEPES-KOH, pH 7.2. Cells were superfused with an extracellular solution containing 140 NaCl, 2.3 NaOH, 1 MgCl 2 , 5.4 KCl, 1.8 CaCl 2 , 5 HEPES, 10 glucose, 2 MnCl 2 , and 1 BaCl 2 , pH 7.4. Currents were recorded and fi ltered on-line at 1 kHz with an Axopatch 200B amplifi er, and acquired using pClamp 9.0 software (Axon Instruments, Inc.). Recordings were performed at 32°C. Activation curves were measured using a two-step protocol in which voltage steps from −25 to −115 or −145 mV were applied from a holding potential of −35 mV, followed by a step to −70 mV. To allow HCN channels to reach steady-state activation at all voltages, the duration of test steps varied from 10 s at −25 mV to 5 s at −145 mV while the duration of the step to −70 mV increased from 8 to 13.5 s after steps from more hyperpolarized voltages. The normalized plot of tail current versus test voltage was fi tted with a Boltzmann function to obtain the V 1/2 and slope factor. Values are given as the mean ± SEM and data were compared using Student's t test for single comparisons and two-way ANOVA followed by a Bonferroni Post-Hoc test for multiple comparisons. Values of P < 0.05 were considered signifi cant.

Reagents and Drugs
An aqueous stock solution of 0.5 mM native PI(4,5)P 2 (Calbiochem) was sonicated and aliquoted before snap freezing in liquid nitrogen. Aliquots were stored at −80°C until the day of the experiment, when working solutions were sonicated a second time. Dioctanoyl (diC 8 ) forms of PI, PI(4)P, PI(3,4)P 2 , PI(4,5)P 2 , and PI(3,4,5)P 3 (Echelon) were dissolved in water to make 2.5 mM stock solutions and stored in aliquots at −80°C. Oleoyl CoA (Sigma-Aldrich) was dissolved in water at a concentration of 5 mM. Anti-PIP 2 mAb and anti-hGM-CSF Ab (Assay Designs) were stored at −20°C at a concentration of 0.909 μg/μl. Antibodies were diluted 1:30 with bath solution to make working solutions. Heat-inactivated anti-PIP 2 mAb was prepared using a 90-min incubation in a 95°C water bath. Wortmannin (Sigma-Aldrich) was dissolved in DMSO at a concentration of 30 mM and stored in aliquots at −20°C. LY294002 (Biomol) was dissolved in DMSO at a concentration of 50 mM and stored in aliquots at −20°C. Polyd-lysine HBr with a molecular weight of 1-4 kD was applied to patches at a fi nal concentration of 25 μg/ml. Materials were purchased from Sigma-Aldrich unless otherwise noted.

Online Supplemental Material
Additional experimental data (available online at http://www. jgp.org/cgi/content/full/jgp.200609648/DC1) show the effect of rundown on the V 1/2 of HCN2 ( Fig. S1) and the effect of phosphatidylinositides on maximal tail current amplitude (Fig. S2).

Exogenous Phosphatidylinositol Phosphates Alter the Voltage-dependent Gating of HCN2 Channels
Hyperpolarization-activated currents were elicited by a series of 3-s hyperpolarizing test pulses in membrane patches from Xenopus oocytes expressing HCN2. The midpoint voltage of activation from tail current activation curves (V 1/2 ) was approximately −80 mV when recorded in cell-attached patches before patch excision or from intact oocytes using two-microelectrode voltage clamp (Chen et al., 2001). However, within 1 min of formation of an inside-out patch, the V 1/2 had shifted by −40 mV, to approximately −120 mV. The V 1/2 was then stable over the next 10 min, indicating that this rundown in the voltage dependence of activation rapidly reached completion (Fig. S1, available at http://www. jgp.org/cgi/content/full/jgp.200609648/DC1).
The role of PI(4,5)P 2 in HCN channel gating was fi rst studied by application of exogenous PI(4,5)P 2 to insideout patches after rundown was complete. Bath applica-tion of 1 μM PI(4,5)P 2 isolated from native tissues, which consists predominantly of phosphatidylinositol 4,5-bisphosphate with an arachidonyl acyl chain in position 2 and a stearyl acyl chain at position 3 (AASt) (Rohacs et al., 2002), produced a large, gradual positive shift in the voltage dependence of HCN2 activation (Fig. 1, A and C). The time course of the shift in V 1/2 upon application of PI(4,5)P 2 was fi t by a single exponential function with a t 1/2 value (time to produce a half maximal effect) of ‫052ف‬ s. The average shift in V 1/2 after 28 min of exposure to phospholipid was +31.8 ± 5.1 mV (n = 4; see Fig. 3 B).
Given the slow rate at which native PI(4,5)P 2 altered the voltage-dependent activation of HCN2, we next examined the short-acyl chain analogue of PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate (diC 8 PI(4,5)P 2 ), since this analogue has been reported to act more rapidly than long chain lipids (Rohacs et al., 2002). Application of 25 μM diC 8 PI(4,5)P 2 caused a signifi cant positive shift in the activation curve of HCN2, which occurred more quickly but was smaller in magnitude than the shift caused by 1 μM AASt PI(4,5)P 2 ( Fig. 1, B and D). The onset of the shift in V 1/2 with diC 8 PI(4,5)P 2 (n = 10) had a t 1/2 value of 86 s, three times faster than the effect of full-length PI(4,5)P 2 (the increased rate of action of diC 8 PI(4,5)P 2 may refl ect the fact that it needed to be applied at a higher concentration than the more potent full-length AASt PI(4,5)P 2 ). The average shift in V 1/2 of HCN2 after 10 min exposure to 25 μM diC 8 PI(4,5)P 2 was +15.4 ± 0.8 mV (n = 12; see Fig. 3 B), and there was a dose-dependent relation between the shift in V 1/2 and the concentration of diC 8 PI(4,5)P 2 (Fig. 2 A). A fi t of the Hill equation to the dose-response curve yielded a value for the maximal shift in V 1/2 of +20.9 mV, a K 1/2 value of 10.9 μM, and a Hill coeffi cient (h) of 1.11.
We next examined the effects of diC 8 PI(4,5)P 2 on the activation and deactivation kinetics of HCN2 channels (Fig. 2 B). Currents during hyperpolarizing voltage steps were fi t with a single exponential to determine an effective time constant of activation. There was no signifi cant effect of PI(4,5)P 2 on the time constant of activation with steps to −115 or −125 mV. However, we observed a signifi cant slowing of activation with steps to −135 mV (P < 0.02; t test). We also observed a significant twofold slowing in the rate of channel deactivation, measured by the time constant of tail current decay upon return of the membrane to −40 mV (P < 0.001; t test).

Specifi city Profi le of HCN2 for Dioctanoyl Phosphatidylinositol Phosphates
Different PI(4,5)P 2 binding domains and PI(4,5)P 2regulated ion channels show varying degrees of specifi city for PI(4,5)P 2 versus phosphatidylinositides that contain different numbers and positions of phosphates on the inositol headgroup (Rohacs et al., 1999;McLaughlin et al., 2002). To determine the lipid specifi city profi le for HCN2, we compared the actions of a variety of PI lipids, all applied at the same concentration (25 μM) (Fig. 3 A).
ATP-sensitive K + channels (K ATP ) are the least specifi c of the inwardly rectifying K + channels in their activation by phosphoinositides and are uniquely activated by long chain (LC) acyl-CoA, an effect that is thought to refl ect this lack of specifi city . Given the low phosphoinositide specifi city of HCN2, we tested the ability of LC acyl-CoA to alter its gating. Bath application of 1 μM Oleoyl CoA caused a +16.0 ± 1.7-mV shift (n = 4) in the HCN2 activation curve that was complete within 2 min. The magnitude of this effect was similar to the shift seen with 25 μM diC 8 PI(4,5)P 2 but signifi cantly smaller than the shift seen with 1 μM native PI(4,5)P 2 (Fig. 3 B). Thus, HCN2 and K ATP appear to defi ne a class of channels that respond to LC acyl-CoA and exhibit a low phosphoinositide selectivity. It is interesting to note that fatty acyl-CoA is a major metabolite of cardiac myocytes, suggesting a possible physiological or pathophysiological role of this modulation.
Interaction of diC 8 PI(4,5)P 2 and cAMP Because the effect of PI(4,5)P 2 to shift the activation of HCN2 to positive voltages is qualitatively similar to the modulatory action of cAMP, we explored the possibility that these two compounds act through similar mechanisms by determining whether cAMP application could occlude the response to diC 8 PI(4,5)P 2 (Fig. 4). Inside-out patches were initially exposed to a saturating concentration of cAMP (10 μM), which resulted in a +15.5 ± 0.6-mV shift in the V 1/2 (n = 4). The subsequent addition of 25 μM diC 8 PI(4,5)P 2 in the continued presence of 10 μM cAMP produced a further shift of +9.5 ± 0.7 mV (n = 4), resulting in a cumulative shift in V 1/2 of +25.0 ± 0.7 mV (Fig. 4 A). Thus, application of a saturating concentration of cAMP did not completely block the effect of diC 8 PI(4,5)P 2 . Nonetheless, the effect of the lipid in the presence of cAMP was signifi cantly less than the 15.4-mV shift in the absence of cAMP (P < 0.01; t test). This suggests that the two compounds bind to distinct sites but may recruit a convergent biophysical mechanism to alter the gating of HCN channels.
We observed a more complex interaction between cAMP and diC 8 PI(4,5)P 2 on activation kinetics. As previously described, cAMP application markedly accelerated HCN2 activation kinetics over a wide range of hyperpolarized voltages (Wainger et al., 2001; Fig. 4 B). In contrast, as mentioned above, diC 8 PI(4,5)P 2 caused a modest slowing of activation kinetics at very negative voltages (−135 mV), with no signifi cant effect on activation kinetics at more positive potentials (see Fig. 2 B). However, when we applied diC 8 PI(4,5)P 2 in the presence of cAMP, the lipid now produced a signifi cant speeding in the rate of channel activation (P < 0.02 at −105 mV and P < 0.001 at −95 mV; t test; Fig. 4 B) at less hyperpolarized voltages. These results reinforce the notion that cAMP and PIP 2 alter channel gating through both shared and unique mechanisms.

cAMP and diC 8 PI(4,5)P 2 Shift HCN2 Activation through Distinct Transduction Mechanisms
To further investigate the relation between the effects of diC 8 PI(4,5)P 2 and cAMP on HCN2 gating, we next examined a mutant HCN2 channel in which a tripeptide sequence-Q450, E451, K452-in the fi rst α helix of the C-linker region (which couples the transmembrane domain to the cyclic nucleotide binding domain) was replaced with a tripeptide sequence, FPN, present at the corresponding region of a cyclic nucleotide-gated channel β subunit. This mutation was previously found to reverse the normal polarity of cyclic nucleotide gating of HCN2 so that cAMP binding shifts voltage-dependent opening of the mutant in the hyperpolarizing direction (Zhou et al., 2004). Thus, if PI(4,5)P 2 acts through a similar molecular mechanism as does cAMP, FPN mutant channels should show a hyperpolarizing response to phospholipid. In fact, we found that diC 8 PI(4,5)P 2 facilitated the opening of HCN2/FPN channels, producing a normal-sized +13.9 ± 1.6-mV shift (n = 4; Fig. 4 A). Thus, PI(4,5)P 2 must regulate HCN2 gating through a transduction mechanism distinct from that used by cAMP, although the effects of the two modulators clearly interact at several different levels.

Rundown of HCN2 Due to Dephosphorylation of Endogenous PIP 2
The above results clearly show that HCN2 is modulated by exogenous PI(4,5)P 2 . We next asked whether HCN2 is also modulated by endogenous PI(4,5)P 2 and whether the rundown upon patch excision may be due to hydrolysis of endogenous PI(4,5)P 2 by membrane-bound lipid Effects of cAMP and diC 8 PI(4,5)P 2 on V 1/2 and channel kinetics are not independent. (A) Interaction of effects of cAMP and diC 8 PI(4,5)P 2 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 + PIP 2 , shift in V 1/2 in response to 10 μM cAMP plus 25 μM diC 8 PI(4,5)P 2 ; PIP 2 (during cAMP), the difference between the shift in V 1/2 in response to cAMP plus diC 8 PI(4,5)P 2 and the shift produced by cAMP alone; PIP 2 , shift in response to 25 μM diC 8 PI(4,5)P 2 alone; PIP 2 (HCN2/FPN), shift of mutant channel in response to 25 μM diC 8 PI(4,5)P 2 . The shift with diC 8 PI(4,5)P 2 in the presence of cAMP is less than the shift with diC 8 PI(4,5)P 2 alone (PIP 2 ) (*, P < 0.01, t test). Error bars show SEM. Number of experiments shown in each bar. (B) Interaction of effects of cAMP and diC 8 PI(4,5)P 2 on time constants (τ) of HCN2 activation (left axis) and deactivation (right axis). Squares, data obtained in absence of PIP 2 and cAMP (solid line, n = 14); circles, data obtained in the presence of 25 μM diC 8 PI(4,5)P 2 (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 diC 8 PI(4,5)P 2 (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 PIP 2 (0.664 ± 0.056 s) was statistically signifi cant (*, 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 PIP 2 (2.18 ± 0.33 s) was also statistically signifi cant (**, P < 0.001, t test).
phosphatases, as observed for other channels Wu et al., 2002).
As a fi rst test of this hypothesis, we examined rundown in the presence of the nonspecifi c phosphatase inhibitors fl uoride (Fl − ) and orthovanadate (VO 4 3− ), which should block or delay any rundown due to lipid dephosphorylation. Patches containing HCN2 were excised into normal bath solution or bath solution supplemented with 0.1 mM Fl − and 0.2 mM VO 4 3− (FV bath solution; Fig. 5). 1 min after patch excision into normal bath solution, the V 1/2 of HCN2 was −120.6 ± 0.5 mV (n = 5). The V 1/2 then remained relatively constant over the next 10 min, indicating that rundown was normally complete within 1 min. However, 1 min after patch excision in FV solution, the V 1/2 was signifi cantly more depolarized than in normal solution (−112.8 ± 0.5 mV; n = 4; P < 0.00001; t test). The V 1/2 then gradually shifted to more hyperpolarized potentials over the next 5-10 min. The V 1/2 for HCN2 in patches excised in FV solution remained signifi cantly positive to the V 1/2 observed in normal bath solution up to 3 min after patch excision (P < 0.04; t test). However, by 5 min after patch excision, the difference was no longer statistically signifi cant. Thus the phosphatase inhibitors fl uoride and orthovanadate delayed but did not prevent HCN2 channel rundown, suggesting that phosphatase activity in the excised patch may contribute to HCN2 channel rundown.
Previous studies suggest that lipid kinases remain associated with the membrane in cell-free patches (e.g., Huang et al., 1998;Rohacs et al., 2002). Thus, as a further test of the hypothesis that endogenous PI(4,5)P 2 contributes to the regulation of HCN2, we next examined whether the effects of rundown on the V 1/2 could be reversed through lipid kinase-mediated phosphorylation. After allowing channel rundown in FV bath solution to reach completion, patches were perfused with FV bath solution plus 2 mM MgATP to promote phosphorylation. Under these conditions, the HCN2 activation curve gradually shifted to more depolarized potentials (Fig. 6 A), reaching an average maximal shift of +9.3 ± 1.4 mV (n = 4) with a t 1/2 of 3.8 min (Fig. 6 B).
This effect of MgATP was due to lipid phosphorylation, as opposed to protein phosphorylation, since we found that the depolarizing shift with MgATP was blocked by the lipid kinase inhibitor wortmannin (15 μM).
In the presence of wortmannin, MgATP produced only a −1.0 ± 1.0 mV (n = 4) shift in the V 1/2 . In the presence of DMSO alone (the vehicle for wortmannin), addition of MgATP shifted the V 1/2 by +7.8 ± 1.1 mV (n = 4) (Fig. 6 B and Fig. 8), which was not signifi cantly different from the MgATP-induced shift in the absence of DMSO (+9.3 mV; P > 0.5; ANOVA). The shifts with MgATP in the absence or presence of DMSO were both signifi cantly greater than the shift in the presence of wortmannin (P < 0.05; ANOVA). 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 signifi cant (*, P < 0.05, t test). For data in response to MgATP alone, the single exponential fi t yielded values for the steady-state shift in V 1/2 of +9.5 mV and a t 1/2 of 3.8 min.
Next we examined the effect of reducing endogenous PI(4,5)P 2 levels by applying to inside-out patches an anti-PI(4,5)P 2 antibody (Huang et al., 1998). After rundown had reached completion in normal solution, application of 30.3 μg/ml anti-PI(4,5)P 2 antibody caused an additional 8.8 ± 1.3 mV hyperpolarizing shift with a time course characterized by a t 1/2 of 3.6 min (Fig. 7 B). Control experiments performed with a heat-inactivated anti-PI(4,5)P 2 antibody or an unrelated antibody failed to produce a signifi cant hyperpolarizing shift (Fig. 8). A further test of the role of endogenous PI(4,5)P 2 came from experiments in which we applied to inside-out patches polylysine, a polycation that reduces the effective negative charge associated with lipid headgroups (Rohacs et al., 2002). Polylysine (25 μg/ml) caused an additional 11.5 ± 1.3-mV hyperpolarizing shift in the HCN2 activation curve (Fig. 8), similar to the effects of the anti-PI(4,5)P 2 antibody, consistent with a regulatory function of endogenous phospholipid.
In addition to manipulating PI(4,5)P 2 levels in insideout patches, we also examined the effects of reducing PI(4,5)P 2 levels in the plasma membrane of intact oocytes. Oocytes were incubated in 15 μM wortmannin for 30-40 min to block the fi nal step in the biosynthetic pathway for PI(4,5)P 2 (Doughman et al., 2003). As a control, other oocytes were incubated in 0.05% DMSO alone. Subsequently, the V 1/2 values for HCN2 in the intact oocytes were assessed under these two conditions by two-microelectrode voltage clamp. Incubation in wortmannin caused a signifi cant hyperpolarizing shift in the V 1/2 of −7.9 ± 1.0 mV relative to the control values (n = 12 pair). As this concentration of wortmannin inhibits both PI-3 kinase and PI-4 kinase, we tested the effects of LY294002, a more specifi c inhibitor of PI-3 kinase (Vlahos et al., 1994;Browaeys-Poly et al., 2000;Song and Ashcroft, 2001). There was no signifi cant effect on the V 1/2 when 20 μM LY294002 was applied to intact oocytes (−0.1 ± 1.9 mV shift relative to DMSO control), indicating a role for PI-4 kinase. Thus, the inhibitory action of wortmannin most likely indicates a role for basal levels of endogenous PI(4,5)P 2 in regulating HCN2 gating in the intact oocyte (Fig. 8).
Regulation of Native HCN Currents in Sinoatrial Node Cells by DiC 8 PI(4,5)P 2 Are native HCN currents in rabbit cardiac sinoatrial cells also regulated by exogenous PI(4,5)P 2 ? To address this question, we evaluated the effect of intracellular dialysis with diC 8 PI(4,5)P 2 on the normal rundown of sinoatrial HCN currents during whole cell recording. The voltage dependence of activation was assayed by a two-step voltage protocol (see Materials and methods), in the absence or in the presence of diC 8 PI(4,5)P 2 (200 μM) in the patch pipette solution (Fig. 9 A). Activation curves determined in the presence or absence of diC 8 PI(4,5)P 2 were identical when measured 1 min after the start of whole-cell recording, with V 1/2 values of −58 mV ( Fig. 9 B). However, after 14 min of whole cell recording, the two activation curves were clearly different. In the absence of diC 8 PI(4,5)P 2 , the V 1/2 shifted to −86 mV, refl ecting rundown. In contrast, with diC 8 PI(4,5)P 2 in the pipette solution, the V 1/2 only shifted to −72 mV (Fig. 9 B). A plot of V 1/2 versus duration of whole-cell recording revealed the progressive hyperpolarizing shift in the HCN activation curve, in both the absence and presence of diC 8 PI(4,5)P 2 (Fig. 9 C). However, diC 8 PI(4,5)P 2 clearly delayed the hyperpolarizing shift, with statistically signifi cant positive shifts observed for the 8-and 14-min time points (P < 0.05).
Comparison of Effects of PIP 2 on HCN2 with Other Channels HCN2 joins a growing list of channels regulated by PI(4,5)P 2 (Suh and Hille, 2005). For some channels, such as TRPV1 (Chuang et al., 2001) and mammalian rod cyclic nucleotide-gated channels (Womack et al., 2000), PI(4,5)P 2 inhibits channel activity. However, for the majority of channels regulated by PI(4,5)P 2 , including HCN2, PI(4,5)P 2 is permissive for channel activity (Hilgemann et al., 2001). Previous studies that investigated the role of PI(4,5)P 2 in channel regulation usually characterized changes in channel function in response to depletion or addition of phosphoinositides to intact membranes. Such studies with the family of inward rectifying K + channels (K ir ) have demonstrated a spectrum of apparent PI(4,5)P 2 affi nities among different channels. Our experiments indicate that HCN2 falls into the K ATP class of channels with relative low specifi city for phosphoinositides.
Although HCN2 showed a relatively low selectivity among the polyphosphoinositides, the channel may have a relatively high affi nity for PI(4,5)P 2 . Channels that tightly bind PI(4,5)P 2 show a very slow response to application of the anti-PI(4,5)P 2 antibody, whereas channels that weakly bind PI(4,5)P 2 are rapidly inhibited by the antibody. For example, the high-affi nity IRK1 channel is inhibited by anti-PI(4,5)P 2 antibody with a half-time of ‫09ف‬ s, whereas the low-affi nity GIRK1/4 and GIRK2 channels show half-maximal block in <10 s (Huang et al., 1998). Despite our having used slightly higher concentrations of antibody than used in the K ir studies, we found that HCN2 required >200 s for halfmaximal inhibition with the anti-PI(4,5)P 2 antibody, suggesting that HCN2 has a high affi nity for PI(4,5)P 2 (although we cannot rule out the possibility that the slow time course of inhibition is due to a particularly slow access of antibody to the inside membrane of the patch in our experiments). A high affi nity of HCN2 for PI(4,5)P 2 is consistent with the idea that this channel constitutively binds PI(4,5)P 2 and that hydrolysis of this phospholipid underlies the ubiquitous rundown of HCN currents.

Interactions of cAMP and PIP 2 : Mechanistic Implications
Our results, unfortunately, do not provide a defi nitive mechanism for how PI(4,5)P 2 regulates HCN2 gating. However, a comparison with the modulatory effects of cAMP provides some interesting clues. The binding of cAMP to its C-terminal cyclic nucleotide binding domain is thought to facilitate HCN channel opening by relieving an inhibitory action of the C terminus on gating (Wainger et al., 2001). Moreover, the effect of cAMP binding to the CNBD is transduced into changes in channel opening through the intervening C-linker region (Chen et al., 2001). We fi nd that a C-linker mutation that reverses the polarity of cAMP action on voltage gating (Zhou et al., 2004) causes no change in PI(4,5)P 2 action, indicating that phospholipid must act through a distinct mechanism from cyclic nucleotide. However, our fi nding that the effects of cAMP and PI(4,5)P 2 are not completely additive suggests that the actions of these modulators may converge on some fi nal mechanistic step of gating, such as the opening of the channel gate at the inner surface of the membrane (Rothberg et al., 2003).

Potential Physiological Implications of Modulation by PIP 2
Our study clearly demonstrates that changes in membrane PI(4,5)P 2 concentration can affect the gating of recombinant HCN2, with higher levels of PI(4,5)P 2 shifting gating to more positive potentials. In addition, we have shown in cells from rabbit sinoatrial node that diC 8 PI(4,5)P 2 also delays the onset of native channel rundown in whole cell recordings. These fi ndings suggest that native HCN channels are regulated by PI(4,5)P 2 and that the presence of this lipid is required to maintain the relatively depolarized range of activation of HCN channels that is required for their opening at physiologically relevant voltages. Moreover, given that rabbit sinoatrial node cells express primarily HCN4 and HCN1 (Shi et al., 1999), our results further indicate that PI(4,5)P 2 is a general modulator of HCN channel function, and does not only target HCN2.
One important remaining question is whether HCN channel function is dynamically regulated in neurons or cardiac myocytes during signal transduction cascades that alter membrane levels of PI(4,5)P 2 . Receptor-mediated activation of phospholipase C results in a decrease in membrane concentration of PI(4,5)P 2 that alters the activity of a number of channels, including GIRK1/4 and KCNQ2/3 (Rohacs et al., 1999;Kobrinsky et al., 2000;Wu et al., 2002;Zhang et al., 2003;Cho et al., 2005). In sinoatrial node cells, a hyperpolarizing shift in HCN channel activation due to reduced levels of PI(4,5)P 2 would slow heart rate, similar to the action of nanomolar concentrations of acetylcholine, which cause a hyperpolarizing shift in pacemaker current without affecting other nodal currents (DiFrancesco et al., 1989). However, such regulation is most effective for channels with a relatively low affi nity for PI(4,5)P 2 , which may or may not hold for HCN channels. In addition to acute regulation by receptor activation, basal levels of PI(4,5)P 2 may also change during development, times of stress, or in disease states (Suchy et al., 1995;Soares et al., 1997;Pasquare et al., 2004;Suh and Hille, 2005); these changes could lead to more tonic changes in HCN channel function. Lastly, PI(4,5)P 2 levels vary among the different membrane compartments of a cell. Thus, a stimulatory effect of PI(4,5)P 2 may allow the cell to restrict channel activity as channels are transported from their site of synthesis in the endoplasmic reticulum to fi nal destinations with more permissive phosphoinositide concentrations (Hilgemann et al., 2001;Suh and Hille, 2005). Further studies will be required to determine the physiological importance of the regulation of HCN channels by PI(4,5)P 2 .