MECHANISM OF BLOCK BY ZD-7288 OF THE HYPERPOLARIZATION-ACTIVATED INWARD RECTIFYING CURRENT IN GUINEA-PIG SUBSTANTIA-NIGRA NEURONS IN-VITRO

1. The effects of the novel bradycardic agent N-ethyl-N-phenylamino)-1 ,2-dimethyl-6-(methylamino pyrimidinium chloride (ZD 7288) (Zeneca) we re investigated on the hyperpolarization-activated cationic current (I -h) in guinea pig substantia nigra pars compacta neurons in vitro, usi ng a single-microelectrode current-clamp/voltage-clamp technique. 2. U nder current-clamp conditions, injection of large negative current pul ses (0.1-0.5 nA, 400 ms) evoked a slow depolarizing ''sag'' in the ele ctrotonic potential due to activation of the slow inward (anomalous) r ectifier. In voltage-clamp recordings, hyperpolarizing voltage steps f rom a holding potential of -60 mV (close to resting potential) elicite d slow inward current relaxations with kinetic properties similar to t hose seen for other neuronal I(h)s. 3. ZD 7288 (10-100 mu M) produced a consistent abolition of the electrotonic potential sag with no effec t on membrane potential or spike properties. Under voltage clamp, I-h amplitude was clearly reduced in a time- and concentration-dependent m anner (apparent half-maximum blocking concentration = 2 mu M); full bl ock of I-h was typically achieved after 10-15 min of exposure to 50 mu M ZD 7288, with no significant recovery observed after 1 h of washing . 4. A similar (although more rapid) block of I-h was seen after appli cation of 3-5 mM Cs+ (partially reversible after 30 min of washing). 5 . Partial block of I-h by 10 mu M ZD 7288 was accompanied by a reducti on in the maximum amplitude of the I-h activation curve, a small negat ive shift in its position on the voltage axis, and a linearization of the steady-state current-voltage relationship. The estimated I-h rever sal potential, however, remained unaffected. 6. In 10 mu M ZD 7288, th e time course of I-h activation and deactivation was significantly slo wed (within the range of -70 to -120 mV for the activation time consta nt and -70 to -90 mV for the inactivation time constant). 7. Blockade of I-h by ZD 7288 or Cs+ was independent of prior I-h activation (i.e. , non-use dependent). 8. Intracellular loading with ZD 7288 also aboli shed the sag in the electrotonic voltage response and I-h relaxations, suggesting an intracellular site of action. By contrast, intracellula r Cs+ had no effect on I-h properties. 9. Block of I-h by ZD 7288 (but not Cs+) was relieved by prolonged cell hyperpolarization, manifested as a slowly developing (half-time similar to 20 s) inward current at a holding potential of -100 mV. 10. We propose that ZD 7288, when appl ied externally, may behave as a ''lipophilic'' quaternary cation, capa ble of passing into the cell interior to block I-h channels in their c losed state; this compound may thus prove a useful research tool, in p lace of Cs+, for studying the properties and significance of I-h curre nts in controlling neuronal function.