Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus

Abstract

The hyperpolarization-activated current (I(h)) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates I(h) conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of I(h) in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. I(h) amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of I(h) to cAMP, and the contribution of the slow component to the overall I(h), decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents.

FIG. 1

Postnatal changes of membrane and I_h properties. (A) Representative examples from P6 (top, RP −69 mV) and P25 (lower traces, RP −74 mV) are shown after current injections (+50 pA to −150 pA). Note the ‘voltage sag’ attributable to I_h activation (asterisk). Scaling: 50 mV, 400 ms. (B) At P22–28 (n = 33, grey), C_M increased and R_M decreased compared with P5–7 (n = 44, black). (C) In voltage-clamp, hyperpolarization (from −63 mV) evoked slow inward currents in P6 (upper traces) and P25 neurons (lower traces). Scaling: 200 pA, 0.5 s. (D) Plotting averaged amplitudes (−133 mV) vs. age shows increasing I_h amplitude (n = 71, 2–13/point). (E) Plotting averaged I_h amplitudes from P5–7 (filled) and P22–28 (open) vs. command potentials shows increased I_h amplitude with hyperpolarization. P22–28 values (n = 21) were significantly higher than P5–7 (n = 20, *P < 0.001) at most potentials. Error bars = SEM.

FIG. 2

I_h activation accelerates during development. (A) Current traces (open symbols) at −133 mV from P6 (upper) and P26 (lower), with superimposed corresponding fits (grey curve). Calibrations 200 pA and 500 ms. (B) τ_slow (open circles) and τ _fast (filled circles) shortened during development (at −133 mV, n = 71, 2–13 cells/point). (C) Averaged τ_fast values from P5–7 (filled squares, n = 20) and P22–28 (open squares, n = 21) were voltage dependent. τ_fast (left) shortened with increasing hyperpolarization, being significantly shorter in P22–28 than P5–7 (P < 0.001, asterisks). Averaged τ_slow values (right) at hyperpolarized potentials were shorter at P22–28 than at P5–7. (D) Average ratios of fast-activating component and total current amplitudes at P5–7 and P22–28 vs. command potentials. At P22–28 the ratio was significantly larger than at P5–7 (*P < 0.001).

FIG. 3

Voltage dependence of activation is significantly shifted to depolarized potentials and is less cAMP-sensitive during development. (A) Activation curves revealed that V_1/2 was shifted in the depolarizing direction at P22–28 (n = 21, open squares) compared with P5–7 (n = 20, filled squares, P < 0.001; insets: tail currents at P6 and P25; scaling: 50 pA, 100 ms). (B) Averaged V_1/2 for each age plotted vs. age, showing a gradual shift of V_1/2 with age (n = 71, 2–10 cells/point). (C) τ_fast,act at potentials close to V_1/2: −103 mV at P5–7 (black bars), −103 mV at P22–28 (bright grey bars; dark grey bars at −93 mV). Statistical significance as compared with P5–7 is indicated by asterisks. (D) V_1/2 was determined in 10 µm cAMP at P5–7 (n = 7, filled squares) and P22–28 (n = 7, open squares). cAMP only affected the activation curve in immature neurons (inset). (E) V_1/2 was determined at P5–7 under control conditions (n = 20) and in 0.01 (n = 8), 10 (n = 7) and 100 µm (n = 4) internal cAMP. V_1/2 shift is already saturated at 10 µm. (F) Hippocampal cAMP levels are age dependent. cAMP was analysed in individual extracts (n = 4/group), under conditions preventing cAMP degradation, by radioimmunoassay. Concentration at P15 was 240% of P6 (*P < 0.001; anova; Bonferroni’s post hoc test) and did not differ significantly between P15 and P23.

FIG. 4

Quantitative analysis of age-dependent mRNA expression of hyperpolarization-activated, cyclic nucleotide-gated channels (HCN)1, 2, 4 channel isoforms in CA1 using ISH (n = 4/group). The figure shows representative autoradiographs and quantitative analysis for each isoform. (A) HCN1 mRNA levels in CA1 (arrowheads) increased progressively with age (P < 0.001). (B) HCN2 mRNA expression tended to increase between P6 and P14, then remained stable, and anova analysis did not show significant changes with age. (C) HCN4 mRNA levels declined as a function of age (P < 0.0001). (D) The relative contribution of HCN channel isoform to the total h-channel mRNA pool was age dependent: HCN4 contribution declined from 28.5 to 14%; HCN1 increased from 36 to 50.6%. *Levels significantly different from P6; †significantly different from P14 (anova, with Bonferroni post hoc tests).

FIG. 5

Protein levels of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN)1, 2 and 4 channel isoforms in hippocampal CA1 vary with age. Protein levels were measured in CA1 samples from individual rats, and were expressed in reference to actin levels in the same samples, as shown. (A). HCN1 protein expression, evaluated using Western blots (n = 4–6/age), correlated well with the mRNA, increasing progressively with age (P < 0.01) as shown in the gel and the quantitative analysis. (B) HCN2 protein levels, analysed using the Alomone antiserum, increased significantly between P6 and P14, then remained stable. (C) HCN4 protein levels decreased significantly with age. (D) Age-dependent contributions of HCN1, HCN2 and HCN4 channel isoforms to total HCN pool of the HCN channels of CA1 cells. For example, the contribution of the HCN1 isoform is predicted to increase from 39 to 65%, and that of HCN4 to decrease from 37 to 4%. *Protein levels significantly different from P6; †significantly different from P14 (P < 0.0001).