Proteolytic processing of HCN2 and co-assembly with HCN4 in the generation of cardiac pacemaker channels

Abstract

In sino-atrial and atrio-ventricular nodal cells, hyperpolarization-activated cyclic nucleotide-gated (HCN) inward current carrying cationic channels, I(f), are expressed that contribute importantly to the diastolic depolarization critical for cardiac pacemaker activity. Although previous studies have demonstrated myocardial expression of both the HCN2 and HCN4 subunits, the specific roles of these subunits in the generation of functional myocardial I(f) channels remain unclear. To explore the molecular compositions of functional cardiac I(f) channels, antibodies targeted against specific C- and N-terminal sequences in HCN2 and HCN4 were exploited to examine HCN2 and HCN4 subunit expression in adult (mouse) heart and to immunoprecipitate endogenous HCN-encoded cardiac I(f) channel complexes. Western blot experiments revealed that although the full-length HCN2 (105 kDa) and HCN4 (160 kDa) proteins are readily detected in transiently transfected HEK-293 cells and in adult (mouse) brain, the molecular mass of the HCN2 protein in the myocardium is approximately 60 kDa. In addition, the myocardial 60-kDa HCN2 protein lacks the C terminus, which contains the cAMP binding domain. In heterologous cells, the C-terminal-truncated HCN2 protein co-assembles with HCN4 to form functional heteromeric HCN channels, which activate faster than homomeric HCN2 or homomeric HCN4 channels, and display properties similar to endogenous myocardial I(f) channels Taken together, these results suggest that functional myocardial I(f) channels reflect the heteromeric assembly of HCN2 and HCN4 subunits and further that the HCN4 subunit underlies the cAMP-mediated regulation of cardiac I(f) channels.

FIGURE 1.

Co-immunoprecipitation of HCN2 and HCN4 from adult (mouse) heart. Proteins were isolated from adult mouse heart and immunoprecipitations (IPs) were performed with the rabbit IgG (control), anti-N-HCN4 (A), or anti-N2-HCN2 (B) polyclonal antibody. Following the immunoprecipitations, proteins were eluted from the beads, fractionated on polyacrylamide (SDS-PAGE) gels, and Western blots (IB) were performed using either the anti-N2-HCN2 (A) or the anti-N-HCN4 (B) polyclonal antibody. Either HCN2 protein bands or HCN4 protein bands were indicated by a closed arrow. IgG bands were indicated by an open arrow. Although full-length (160 kDa) HCN4 protein was readily identified in the heart lysate, i.e. prior to the immunoprecipitation (lane 4) and in the (immunoprecipitated) protein sample obtained following the immunoprecipitation with the anti-N2-HCN2 antibody (B), the anti-N2-HCN2 antibody detected only a single low molecular mass ∼60-kDA protein (A) in both the immunoprecipitated protein sample and in the Western blot of the whole heart lysate. No additional HCN2 (A) or HCN4 (B) proteins were observed in the samples obtained following further washing of the beads (lanes A3 and B3). As is also evident, neither HCN2 (A) nor HCN4 (B) was immunoprecipitated using rabbit IgG (lanes A1 and B1).

FIGURE 2.

Post-translational modification at the C terminus of the HCN2 protein in the heart. Proteins isolated from HEK-293 cells transiently transfected with (full-length) HCN2 and from mouse brain or heart were fractionated and immunoblotted (IB) with the anti-N1-HCN2 (A), anti-N2-HCN2 (B), anti-C1-HCN2 (C), or anti-C2-HCN2 (D) antibody, as indicated. HCN2 protein bands were indicated by a closed arrow. As expected, the full-length (∼105 kDa) HCN2 protein was readily and reliably detected in the samples prepared from the HCN2-expressing HEK-293 cells. In addition, a single ∼105-kDa band was identified in brain. In contrast, only a much lower molecular mass, ∼60-kDa band was detected in the heart protein samples using either the anti-N1-HCN2 or the anti-N2-HCN2 antibody. In addition, there were no proteins identified in the heart samples using either the anti-C1-HCN2 or the anti-C2-HCN2 antibody, although both antibodies detected the full-length HCN2 protein in brain and in HCN2-expressing HEK-293 cells.

FIGURE 3.

Detection of full-length channel proteins in heart lysates. Proteins, isolated from HEK-293 cells stably expressing HCN4 and from adult mouse heart, were fractionated on SDS-PAGE gels and immunoblotted (IB) with the anti-N-HCN4 antibody (A) or the anti-C-HCN4 antibody (B). HCN4 and Nav1.5 protein bands were indicated by a closed arrow. A ∼160-kDa band was detected in samples from the HEK-293 cell line stably expressing HCN4 and in mouse heart. Nothing was detected in untransfected (wild type) HEK-293 cells with either of these (anti-HCN4) antibodies. C, similarly, a single, high molecular mass protein (at ∼250 kDa) was detected in lysates from adult mouse heart and from HEK-293 cells stably expressing Nav1.5, but not in extracts from untransfected HEK-293 cells.

FIGURE 4.

C-terminal modification of HCN2 does not reflect proteolysis during protein isolation and/or handling. Whole cell lysates were prepared from HEK-293 cells transfected with a cDNA construct encoding an HA-tagged HCN2, and Western blots of these samples and of heart lysates were probed for HCN2 expression. HCN2 protein bands were indicated by a closed arrow. A, using the anti-HA antibody, only the full-length (HA-tagged) HCN2 protein was detected in lysates from HEK-293 cells (lane 3), as well as in the sample prepared by mixing the heart and the HEK-293 cell lysates (lane 2). In contrast, and as expected, nothing is detected with this antibody in the heart lysates (lane 1) or in lysates from untransfected HEK-293 cells (lane 4). Following stripping, the membrane was re-probed with the anti-N2-HCN2 antibody (B). As is evident, the full-length HCN2 and the 60-kDa HCN2 proteins were detected in the sample (lane 2) prepared by mixing the heart lysate with the extract from HCN2-expressing HEK-293 cells, whereas only the full-length HCN2 is detected in HEK-293 cells (lane 3), and only the 60-kDa protein is detected in the heart (lane 1).

FIGURE 5.

A C-terminal-truncated HCN2 (HCN2ΔC) protein forms heteromeric channels with HCN4 in HEK-293 cells. A, representative recordings obtained from (wild type or HCN4) expressing HEK-293 cells following transient transfection with cDNA constructs encoding the full-length HCN2 or the C-terminal HCN2 truncation in mutant, HCN2ΔC. Representative currents in HEK-293 cells stably expressing HCN4 are also illustrated. In each case, currents evoked during 3 s hyperpolarizing voltage steps to test potentials between −130 and −60 mV from a holding potential of −40 mV are illustrated; the voltage clamp paradigm is illustrated below the records. B, peak currents at each test potential in each cell were measured and normalized to the maximal peak current recorded in the same cell, and mean ± S.E. normalized peak currents are plotted as a function of the test potential; the individual curves were fitted to the Boltzmann equation (see “Materials and Methods”). Mean ± S.E. voltages (V_1/2) of half-maximal current activation (C) and activation time constants (D) are illustrated; n values are presented above the bars (*, p < 0.05).