Primary structure of the human HCN4 channel and sequence comparison with mouse subtypes mHCN1–mHCN3. Identical residues with respect to hHCN4 sequence are represented by empty spaces and gaps by dashes. The six predicted transmembrane segments (S1–S6), the pore region, and the cyclic nucleotide-binding domain are indicated by bars above the sequence. Sequences of mHCN1–mHCN3 are from ref. 9.

Sections of human metaphase spreads after in situ hybridization with a biotinylated probe of hHCN4, detected with FITC conjugated to avidin. Arrows indicate the fluorescent signal on chromosome region 15q24–q25. Chromosomes were counterstained with DAPI.

(A) Current responses to hyperpolarizing voltage steps to −120 mV for 1 s and tail currents recorded by stepping the voltage to the indicated value (see voltage protocol above the current traces). The arrow indicates the instantaneous tail-current amplitudes used for the I/V_m relation shown in B. (B) Instantaneous I/V_m relation derived from tail currents in A. The reversal potential was −22 mV.

Northern blot analysis of hHCN4 gene expression. Each lane contains 2 μg of poly(A)⁺ RNA from each of the following human tissues: He, heart; Br, total brain; Pl, placenta; Lu, lung; Li, liver; Mu, skeletal muscle; Ki, kidney; Pa, pancreas (A); Sp, spleen; Thy, thymus; Pr, prostata; Te, testis; Ov, ovary; In, small intestine; Co, colon; Leu, peripheral blood leukocytes (B); Am, amygdala; CN, caudate nucleus; CC, corpus callosum; Hi, hippocampus; Br, total brain; SN, substantia nigra; Th, thalamus (C); and Ce, cerebellum; CeC, cerebral cortex; Me, medulla; SC, spinal cord; OP, occipital pole; FL, frontal lobe; TL, temporal lobe; Pu, putamen (D). Blots were hybridized with a ³²P-labeled 267-bp cDNA fragment.

Functional characterization of hHCN4. (A) Whole-cell current responses to hyperpolarizing voltage steps from a holding potential of +10 mV to test values between −50 and −150 mV in increments of 10 mV. Tail currents were recorded by stepping the voltage back to +10 mV. The arrow indicates the instantaneous tail current. (B) Voltage dependence of the activation rates. Time constants τ (±SD, ●) of current activation were determined by fitting single exponentials to the current traces of recordings as in A (−130 to −150 mV) and in C (all other voltages). The number of experiments is given in parentheses. Time constants of activation of native I_h currents (▴) recorded from thalamocortical neurons were taken from ref. 11; ○ represent time constants for hHCN4 at 36°C, calculated from the data at 21°C, assuming a Q₁₀ = 6. (C) Whole-cell current responses to hyperpolarizing voltage steps (see protocol above the current traces) of 7,500 ms duration (gray traces) and superimposed fit with a single exponential function (solid lines). The Inset shows tail currents recorded at +50 mV on an expanded time scale. (D) Voltage dependence of instantaneous tail currents (●), taken from C (Inset , arrow), representing the relative P_o. Solid line is a fit of the Boltzmann function (I − I_min)/(I_max − I_min) = [1 − exp([V − V_1/2]/s)]⁻¹ with V_1/2 = −79.6 mV and s = 7.9 to the data. (E) Normalized tail currents (symbols) and fits (solid lines) of experiments with varying duration t_p of the hyperpolarizing pulse: ■, t_p = 300 ms, V_1/2 = −151.4 mV; ○, t_p = 1 s, V_1/2 = −119.0 mV; ●, t_p = 7.5 s, V_1/2 = −85.2 mV; ▴, t_p = 60 s, V_1/2 = −75.2 mV. (F) Mean values ± SD for V_1/2 (determined as in D and E) plotted against duration of the hyperpolarizing pulse. V_1/2 saturates for long pulse durations (>60 s) at −75.2 (±3.4) mV.

Modulation of hHCN4-mediated currents by cAMP. (A) Incremental increase of steady-state current at −100 mV after photorelease of cAMP from caged cAMP. (Inset) Time course of flash-induced current increase displayed on a larger time scale. (B) Comparison of time courses of hHCN4 currents activated by steps to −120 mV before and after release of cAMP by the UV flash. (C) Voltage dependence of relative P_o derived from tail current analysis similar to that shown in Fig. 4 C and D in the absence (○) and presence (●) of cAMP. Midpoint potentials (V_1/2) and slope factors (s) were determined from a fit of the Boltzmann equation to the data. Mean V_1/2 (control) = −85.2 ± 5.6 mV; s = 9.2 ± 1.9 mV (13 experiments); mean V_1/2 (cAMP) = −74.1 ± 5.3 mV; s = 9.7 ± 2.1 mV (10 experiments). (D) Demonstration of open hHCN4 channels at a depolarized potential (−40 mV). From a holding voltage of −40 mV, short voltage pulses of 10-ms duration to +50 mV were repetetively applied every 10 s. After 100 s, a flash of UV light (500 ms) was applied, releasing cAMP from the caged compound. Three example traces are shown at t = 100 s, t = 140 s, and t = 550 s. Capacitive current transients have been removed for clarity. (E) Rise and decay of flash-induced changes in current amplitude measured at +50 mV in D.