Modulation of ion channels by metabotropic transmitters and receptors is a widespread mechanism for the regulation of excitability in the brain. These mechanisms are slow compared with ionotropic receptors but the speed of modulation is difficult to discern independently of the agonist-receptor interaction in intact tissue. We used photolysis of caged cAMP to reveal the time course of the cAMP-dependent modulation of the Ca²⁺-dependent K⁺ current I_sAHP in hippocampal neurones.

Hippocampal slices were obtained from humanely killed 16- to 21-day-old rats. Whole-cell recordings were made from CA1 pyramidal cells using KMeSO₄-based solutions plus DMNB-caged cAMP. Prolonged photolysis of caged cAMP was produced by a xenon lamp and UV filter on an upright microscope. Rapid photolysis was achieved with a flashlamp. I_sAHP was evoked either by depolarising voltage steps or internal perfusion with 1 mM dibromo-BAPTA (DBB, Lancaster & Batchelor, 2000) to produce a persistently active I_sAHP. Data are expressed as means ± S.E.M. sAHP charge was measured as 1 s integrals of the current commencing 500 ms from the end of the depolarising step.

Flash photolysis of 200 µM caged cAMP (1 ms flash discharge) caused almost complete inhibition of depolarization-evoked I_sAHP (94 ± 9 %, n = 4). In contrast, prolonged photolysis (30 s UV) of 200 µM caged cAMP was less effective and inhibited I_sAHP by 56 ± 10 % (n = 4). Similar prolonged photolysis with the persistently active I_sAHP showed that inhibition became evident 2-3 s after the onset of UV exposure and reached a steady state of 43 ± 5 % (n = 5) after 20 s. The sAHP charge was also inhibited by bath application of the phosphodiesterase (PDE) inhibitor IBMX (1 mM; 105 ± 27 to 23 ± 12 pC, n = 5). This is consistent with tonic PDE activity which restrains accumulation of cAMP. Further definition of the onset of inhibition was obtained using a flashlamp linked to a 700 µm fibre-optic bundle. The fibre-optic tip was placed through the solution interface adjacent to the tissue, so minimising inter-experiment variability. Photolysis of increasing concentrations of caged cAMP (40, 200 and 1000 µM; n = 5 in each case) caused progressively greater and more rapid inhibition of the K⁺ current with time constants of 4229 ± 638 ms (40 µM), 2213 ± 183 ms (200 µM) and 1650 ± 186 ms (1000 µM).

These data indicate some of the temporal limits of cAMP-dependent regulation of neuronal ion channels.

This work was supported by The Wellcome Trust, Sir Jules Thorn Charitable Trust and Norwegian Research Council.