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 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino pyrimidinium chloride (ZD 7288) (Zeneca) were investigated on the hyperpolarization-activated cationic current (I-h) in guinea pig substantia nigra pars compacta neurons in vitro, using a single-microelectrode current-clamp/voltage-clamp technique. 2. Under current-clamp conditions, injection of large negative current pulses (0.1-0.5 nA, 400 ms) evoked a slow depolarizing ''sag'' in the electrotonic potential due to activation of the slow inward (anomalous) rectifier. In voltage-clamp recordings, hyperpolarizing voltage steps from a holding potential of -60 mV (close to resting potential) elicited slow inward current relaxations with kinetic properties similar to those 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 effect on membrane potential or spike properties. Under voltage clamp, I-h amplitude was clearly reduced in a time- and concentration-dependent manner (apparent half-maximum blocking concentration = 2 mu M); full block 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 application 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 reduction in the maximum amplitude of the I-h activation curve, a small negative shift in its position on the voltage axis, and a linearization of the steady-state current-voltage relationship. The estimated I-h reversal potential, however, remained unaffected. 6. In 10 mu M ZD 7288, the time course of I-h activation and deactivation was significantly slowed (within the range of -70 to -120 mV for the activation time constant 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 abolished the sag in the electrotonic voltage response and I-h relaxations, suggesting an intracellular site of action. By contrast, intracellular 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 applied externally, may behave as a ''lipophilic'' quaternary cation, capable of passing into the cell interior to block I-h channels in their closed state; this compound may thus prove a useful research tool, in place of Cs+, for studying the properties and significance of I-h currents in controlling neuronal function.