Cyclic AMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones

1. Bull-frog sympathetic neurones in primary culture were voltage clamped in the whole-cell configuration. The pipette solution contained ATP (5 mM). 2. A hyperpolarization-activated sodium-potassium current (H-current: IH) was separated from other membrane currents in a nominally calcium-free solution containing cobalt (2 mM), magnesium (4 mM), barium (2 mM), tetraethylammonium (20 mM), tetrodotoxin (3 microM), apamin (30 nM) and 4-aminopyridine (1 mM). IH was selectively blocked by caesium (10-300 microM). 3. The steady-state activation of IH occurred between -60 and -130 mV. The H-conductance was 4.1-6.6 nS at the half-activation voltage of -90 mV. With the concentrations of potassium and sodium ions in the superfusate at 20 and 70 mM, respectively, the reversal potential of IH was about -20 mV. IH was activated with a time constant of 2.8 s at -90 mV and 22 degrees C. The Q10 between 16 and 26 degrees C was 4.3. 4. A non-hydrolysable ATP analogue in the pipette solution did not support IH activation. Intracellular 'loading' of GTP-gamma-S (30-500 microM) led to a progressive activation of IH. 5. Forskolin (10 microM) increased the maximum conductance of IH by 70%. This was associated with a depolarizing shift in the half-activation voltage (5-10 mV) and in the voltage dependence of the activation/deactivation time constant of IH. 6. Essentially the same results as with forskolin were obtained by intracellular 'loading' with cyclic AMP (3-10 microM) or bath application of 8-bromo cyclic AMP (0.1-1 mM), dibutyryl cyclic AMP (1 mM) and 3-isobutyl-1-methylxanthine (0.1-1 mM). 7. The protein kinase inhibitor H-8 (1-10 microM) decreased the peak amplitude of IH. Phorbol 12-myristate 13-acetate (10 microM), a protein kinase C activator, was without effect. 8. It is concluded that a voltage-dependent cation current can be regulated by the basal activity of adenylate cyclase, presumably through protein kinase A, in vertebrate sympathetic neurones.