No abstract

Ih is a poorly selective cation current that activates upon hyperpolarization, present in various types of neurons. Our aim was to perform a detailed thermodynamic analysis of Ih gating kinetics, in order to assess putative structural changes associated with its activation and deactivation. To select dorsal root ganglia neurons that exhibit large Ih, we applied a current signature method by Petruska et al. (J Neurophysiol 84:2365-2379, 2000) and found appropriate neurons in cluster 4. Currents elicited by 3,000-ms hyperpolarizing pulses at 25 and 33 degrees C were fitted with double exponential functions, yielding time constants similar to those of HCN1. The fast activation and deactivation rates showed temperature coefficients (Q10) of 2.9 and 3.1, respectively, while Q10 of the absolute conductance was 1.3. Using the Arrhenius-Eyring formalism we computed heights of voltage-independent Gibbs free energy and entropy barriers for each rate. The free energy barriers of the fast rates were just approximately 2RT units lower than those of the corresponding slow rates (31.3 vs. 33.2RT for activation, and 24.7 vs. 25.8RT for deactivation, at 25 degrees C). Interestingly, the entropy barriers of the slow rates were negative: -15.2R units for activation and -11.9R units for deactivation, compared to 4.6 and 1.3R units, respectively, for the fast component. The equivalent gating charge (zg) (3.75 +/- 0.32, mean +/- SEM, at 25 degrees C) and half-activation potential (V1/2) (-70.0 +/- 1.3 mV at 25 degrees C) did not vary significantly with temperature.

This study was undertaken with the aim of testing the action of amitriptyline on the epithelial Na channel (ENaC), which belongs to the same family (Deg/ENaC) as ASICs (acid-sensing ion channels) and many other putative members in the brain. We assumed that, having a common protein structure, characterization of the amitriptyline-ENaC interaction could help to elucidate the analgesic mechanism of this tricyclic antidepressant. Na-channel characteristics were derived from the analysis of blocker-induced lorentzian noise produced by amiloride. The effect of amitriptyline, present in the mucosal bathing solution, on the transepithelial short-circuit current (I(sc)) and conductance (G(t)), and on the blocker-induced noise of apical Na channels, was studied on isolated ventral skin of the frog Rana ridibunda. Amitriptyline exerted a dual effect on the macroscopic short-circuit current and conductance of the epithelia, increasing these two parameters in the concentration range 0.1-50 microM, while at higher concentrations (100-1000 microM) it showed an inhibitory action. The decrease in the association rate (k(01)) of amiloride to the apical Na channels from 15.6+/-4.2 microM(-1) s(-1) in control Cl-Ringer to 7.4+/-1.7 microM(-1) s(-1) at 200 microM amitriptyline in a concentration-dependent manner suggests a competitive binding of amitriptyline to the pyrazine ring binding site for amiloride.

Analgesia induced by certain tricyclic antidepressants has been largely used for decades, yet the mechanisms involved are incompletely understood. Starting from previously reported dual effects of amitriptyline on wild-type ENaC (see ref. 39), we extended our study to ASIC1a by performing a series of whole cell and singlechannel recordings of proton-activated currents in HEK293 cells. Acid pulses were applied at 2 or 5 min intervals, and amitriptyline (1-500 μM) was applied at a holding pH of 7.4 or 8.4 between pulses. Dose-response plots were fitted with dual Hill type functions, yielding a half-activatory constant of 0.3 μM and a half-inhibitory constant of 382 μM at pH 7.4. At pH 8.4 both constants were shifted to higher values (0.5 and 444 μM, respectively). In whole-cell experiments, FMRF-amide increased the peak amplitude of ASIC1a transients at 0.1 μM and decreased it at 1 and 100 μM. Single-channel recordings were idealized and fitted using an 8-state linear connectivity model comprising four consecutive activation steps. Both amitriptyline (1 μM) and FMRF-amide (0.1 μM) increased the unitary current amplitude, and modified the opening and closing rates of the first gating mode. They also increased the transition rate from the second to the first gating mode, and the rate of final closure. The activatory effect of both compounds vanished after a mild trypsin pretreatment, suggesting the existence of activatory sites for FMRF-amide and amitriptyline in the outer vestibule of ASIC1a, which can be removed by exo-or endogenous serine-proteases.