Heteromeric HCN1-HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node

Abstract

'Funny-' (f-) channels of cardiac sino-atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f-channels are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f-channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co-assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co-transfected (HCN4 + HCN1) and concatenated (HCN4-HCN1 (4-1) tandem or HCN1-HCN4 (1-4) tandem) rabbit constructs and compared them with those of the native f-current from rabbit SAN. 4-1 tandem, but not co-transfected, currents had activation kinetics approaching those of If; however, the activation range of 4-1 tandem channels was more negative than that of the f-channel and their cAMP sensitivity were poorer (although that of 1-4 tandem channels was normal). Co-transfection of 4-1 tandem channels with minK-related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f-channels, but a 'context'-dependent mechanism is also likely to modulate the channel properties in native tissues.

Figure 1. Co-expression of HCN1 and HCN4 proteins (A and B) and of GFP and MiRP1 proteins (C and D) in HEK 293 cells

A and C, Western blot analyses performed on membrane proteins (50 μg per lane) from HEK 293 cells co-transfected with HCN1 + HCN4 cDNA (A) or transfected with a polycistronic vector carrying MiRP1 and GFP cDNAs (C), using polyclonal anti-HCN1 (A, left), anti-HCN4 (A, right) or anti-MiRP1 antibodies (C). Lack of primary antibody exposure (A) or mock cell transfection (C) yielded no signal (not shown). For each of the three proteins, the molecular masses obtained were close to those expected (HCN1, Demontis et al. 2002; HCN4 and MiRP1, Alomone Labs datasheets). B and D, immunolocalization of HCN1, HCN4 or MiRP1 in HEK 293 cells. In B, cells were co-transfected with HCN1 +HCN4 and treated with either anti-HCN1 (upper left) or anti-HCN4 antibodies (lower left), as indicated. Panels on the right show phase contrast images. These confocal microscopy images were obtained from the superimposition of 9 sections spanning 5.86 μm (B, upper left), of 5 sections spanning 2.24 μm (B, lower left) and of 5 sections spanning 4.25 μm (D). In D, MiRP1 (upper left) and GFP(upper right) signals are shown in the same cell. The two images are overlapped in the bottom panel.

Figure 3. Comparison between activation time constants of HCN currents and of native I_f

A, sample traces recorded on hyperpolarization to the voltages indicated; at each voltage, the step duration was long enough for steady-state activation to be reached. B, activation time constants; data were collected from single exponential fits of activation time courses (see Methods) from a total of n = 4, 21 and 15 cells expressing HCN1, HCN4 and co-transfected HCN1 + HCN4 channels, respectively and from n = 20 SAN cells. Curves drawn through points.

Figure 2. Comparison between activation time courses of HCN currents and of native I_f

All traces were recorded during hyperpolarizing steps from a holding potential of −35 mV to −105 mV and are clipped to 2 s to improve clarity. In all panels, the same I_f trace recorded from a SAN cell is shown (right-hand axes). A-C, comparison of I_f with currents recorded from HEK 293 cells expressing HCN1 alone, HCN4 alone and co-transfected HCN1+HCN4, respectively, as indicated (left-hand axes). I_f activated more slowly than HCN1 and more rapidly than HCN4 or co-transfected HCN1+HCN4 currents.

Figure 4. Comparison between activation curves of HCN and of native f-channels

A, sample traces recorded at −125 (f), −125 (HCN1), −145 (HCN4) and +5 mV (HCN1 + HCN4) following current activation at the voltages indicated. For each trace, activation step duration was selected to allow attainment of steady state (see Fig. 3). B, fractional activation curves; data points were obtained from a total of n = 5, 7 and 11 cells expressing HCN1, HCN4 and co-transfected HCN1 + HCN4 channels, respectively and from n = 5 SAN cells. Fitting of data by the Boltzmann equation (continuous lines) yielded half-activation voltages (V_1/2) of −73.0, −79.5, −82.9 and −62.5 mV and inverse slope factors (s) of 8.7, 10.2, 10.6 and 9.6 mV for the HCN1, HCN4, HCN1 + HCN4 and f curves, respectively.

Figure 5. Kinetic properties of 4–1 tandem channels compared to those of individual isoforms, co-transfected HCN1 + HCN4 and native channels

A, sample 4–1 tandem trace recorded during a step from −35 to −100 mV, compared to HCN1-, HCN4- and f-current traces recorded in similar conditions (same records as in Fig. 2), as indicated. Traces are clipped to 2 s for clarity. The 4–1 tandem record approximates the I_f record. B, voltage dependence of the time constant curve of 4–1 tandem current activation; means ±s.e.m. values were averaged from n = 14 cells. Also plotted as continuous lines (only lines through points) are the curves for HCN1-, HCN4-, HCN1 + HCN4- and f-channels (as in Fig. 3B), as indicated. The 4–1 tandem trace approaches the native f-channel trace, but tandem time constants were larger at all voltages. C, activation curve of 4–1 tandem current, averaged from n = 14 cells (mean ±s.e.m.); fitting parameters for Boltzmann curve (dashed line) are given in text. Also plotted are Boltzmann-fitted curves for HCN1-, HCN4-, HCN1 + HCN4- and f-channels (continuous lines, from Fig. 4B).

Figure 6. Responses of HCN and native f-channels to cAMP

A-F, representative current records during hyperpolarizing steps from a holding potential of −35 mV for I_f, HCN1, HCN4, co-transfected HCN1 + HCN4, 4–1 tandem and 1–4 tandem channels, respectively; traces were recorded in control conditions, during cAMP application (*) and after return to control as indicated. In all cases except for HCN1, 10 μm cAMP was perfused on the intracellular side of inside-out patches; HCN1 traces are whole-cell recordings taken before, during and after extracellular perfusion with 100 μm pCPT-cAMP. Step voltages and cAMP-induced shifts were (mV): −95, 12.2 (A); −90, 7.5 (B); −105, 17.5 (C); −95, 13.5 (D); −95, 6.6 (E); and −115, 10.7 (F). G, bar graph of responses to 10 μm cAMP (or 100 μm pCPT-cAMP in the case of HCN1) measured as shifts in the activation curve (see Methods for details) during protocols as in A-F. Mean ±s.e.m. values and number of cells used are reported in the text.

Figure 7. Lack of action of MiRP1 on the properties of HCN4 channels

A, representative traces recorded during steps to −95, −105 and −115 mV from a holding potential of −35 mV from an HEK 293 cell expressing HCN4 alone (left) or in combination with MiRP1 (right), as indicated. The time course is similar at all voltages. B and C, time constant curves (B) and activation curve s (C) for HCN4 alone (•) and HCN4+MiRP1 (□). Means ±s.e.m. plotted from n = 5 cells expressing HCN4 and n = 4 cells expressing HCN4 + MiRP1. In B, lines are drawn through points. In C, lines are Boltzmann fits to data points (fitting values in text).

Figure 8. Lack of action of MiRP1 on the properties of 4–1 tandem channels

A, representative traces recorded during steps to −95, −105 and −115 mV from a holding potential of −35 mV from a HEK 293 cell expressing 4–1 tandem channels alone (left) or in combination with MiRP1 (right), as indicated. B and C, time constant curves (B) and activation curve s (C) for 4–1 tandem channels alone (•) and in combination with MiRP1 (□). Mean ±s.e.m. values were plotted from n = 6 cells expressing 4–1 tandem channels alone and n = 5 cells expressing 4–1 tandem channels + MiRP1. In B, lines are drawn through points. In C, lines are Boltzmann fits to data points (fitting values in text).