Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node

Abstract

The sympathetic nervous system increases heart rate by activating beta adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (I(f)) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. I(f) is produced by hyperpolarization-activated cyclic nucleotide-sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. beta Adrenergic regulation of I(f) in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of beta adrenergic agonists to shift the voltage dependence of I(f) in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of I(f). Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate.

Figure 1.

PKA activity contributes to β adrenergic regulation of I_f in sinoatrial myocytes. (A and C) Representative whole cell I_f currents recorded from isolated sinoatrial myocytes in the whole cell patch clamp configuration. Currents were elicited by voltage steps to −120 mV from a holding potential of −50 mV in the absence (black) or presence (red or blue) of 1 µM isoproterenol (ISO) applied in the extracellular Tyrode’s solution. Traces in A were recorded with a control intracellular solution, traces in C were recorded with intracellular solution containing 10 µM PKI. (B and D) Time course of the ISO-dependent increase in I_f for the cells shown in A and C. The open and colored bars indicate the composition of the extracellular perfusing solution. Black, red, and blue circles mark the traces shown in A and C.

Figure 2.

PKA inhibition reduces β adrenergic regulation of I_f in sinoatrial myocytes. (A and B) Conductance–voltage relationships for I_f determined in control intracellular solution (A) or intracellular containing 10 µM PKI (B) in the absence (closed black or gray symbols) and presence (open red or blue symbols) of 1 µM isoproterenol. (A, inset) Representative currents elicited by 3-s hyperpolarizing voltage steps from −60 to −170 mV in 10-mV increments. Scale bars in the inset are 500 pA and 200 ms. (C) Average activation midpoints for I_f in different conditions, as indicated. Isoproterenol significantly shifted the voltage dependence for I_f in the absence of PKI (asterisk, P < 0.05), but not in the presence of PKI (NS, not significant).

Figure 3.

PKA shifts the voltage dependence of HCN4 in CHO cells. (A) Average voltage dependence of activation for HCN4 currents. Normalized tail current amplitudes are plotted as a function of prepulse voltage for protocols as shown in the inset with control intracellular solution (filled black circles), intracellular with 20 U/ml PKA (open red circles), or intracellular with PKA plus 10 µM PKI (open blue triangles). Midpoint activation voltages were −89.8 ± 1.7 mV in control, n = 7; −83.8 ± 1.5 mV in PKA, n = 9; and −90.0 ± 2.0 mV in PKA + PKI, n = 8. (Inset) Representative whole cell currents recorded from a CHO cell line stably expressing HCN4. Currents were elicited by hyperpolarizing voltage steps from −40 to −150 mV (in 10-mV increments), which ranged in length from 32.5 to 4.2 s (starting with the longest step at −40 mV and decremented by 2.575 s each step). This voltage protocol was used in an attempt to measure the steady-state voltage dependence of activation; however, note that activation was still incomplete at the more depolarized potentials. (B) Activation midpoints for HCN4 currents from Boltzmann fits to data in A. Asterisk indicates statistical significance (P < 0.05). NS, not significant (P > 0.05).

Figure 4.

PKA phosphorylates HCN4 channel fragments in vitro. (A) Schematic representation of HCN4 GST fusion proteins. (B) In vitro phosphorylation and Western blotting of HCN4 channel fragments fused to GST. Fusion proteins were incubated with ³²P-ATP in the absence or presence of PKA (20 U/ml) or PKA plus PKI (20 µM) as indicated. Incorporated ³²P was visualized by autoradiography (top), and GST fusion proteins were visualized by Western blotting with an anti-GST antibody (1:50,000; Oncogene; bottom). (C) Average densitometric analysis of ³²P incorporation into the HCN4 GST fusion proteins for three independent experiments. Red bars show average density of ³²P normalized to the density of corresponding protein bands in the presence of PKA, blue bars, in PKA plus PKI. Asterisks indicate a significant reduction in normalized ³²P incorporation in the presence of PKI.

Figure 5.

Identification of PKA-phosphorylated residues in HCN4 GST fusion proteins. Compiled peptide sequences from tandem MS analysis of the HCN4 portions of the GST fusion proteins. Bold underlined residues were identified by MS sequencing; plain text residues were not resolved by MS. Red highlighting indicates residues found to be phosphorylated in multiple peptide fragments, and green highlighting indicates residues found to be phosphorylated in single peptide fragments.

Figure 6.

Comparison of PKA-phosphorylated residues on HCN4 to predicted PKA phosphorylation sites. Amino acid sequence of mHCN4 is shown with color coding to illustrate phosphorylated residues identified by MS compared with predicted PKA sites. Red residues were phosphorylated in multiple peptide fragments, and green residues in single peptide fragments. Sequences identified as high probability PKA phosphorylation sites (score ≥ 0.5) by the pkaPS algorithm are highlighted in yellow. The cyclic nucleotide binding domain is indicated by a black outline, and transmembrane regions by gray shading. (Inset) Schematic representation of an HCN4 subunit with the approximate location of identified phosphorylated residues marked by red and green circles. The cyclic nucleotide binding domain is represented by a thick black line.

Figure 7.

A regulatory site in the distal C terminus of HCN4 is required for PKA modulation. Representative whole-cell current traces (insets) and average conductance–voltage plots for wild-type HCN4 (A), HCN4-ΔNΔC (B), HCN4-ΔC (C), HCN4-Nx4 (D), HCN4-Δ1012 (E), and HCN4-Cx4 (F) expressed in CHO cells. Black lines and closed circles indicate control intracellular solution; red lines and open circles indicate PKA-containing intracellular solution. Currents were elicited by 3-s hyperpolarizing voltage steps ranging from −40 to −180 mV in 10-mV increments (as indicated). Scale bars: (A, B, and E, insets) 500 pA, 500 ms; (C, D, and F, insets) 1 nA, 500 ms. Cartoons in each panel depict the constructs, where red and green circles indicate confirmed and unconfirmed PKA sites, and open circles indicate alanine substitutions of PKA sites.

Figure 8.

Summary of PKA-dependent shifts in V_1/2 for HCN4 constructs. Average activation midpoints from Bolztmann fits for wtHCN4, HCN4-ΔNΔC, and HCN4-ΔC in the absence (black bars) or presence (red bars) of 20 U/ml PKA in the intracellular pipette solution. Asterisks indicate statistical significance (P < 0.05) between control and PKA intracellular solution. Double daggers indicate significant differences from wtHCN4 in the absence of PKA.