On the mechanism of block by Zeneca ZD 7288 of the hyperpolarization- activated inward rectifying current ( I h ) in guinea-pig substantia nigra neurones in vitro. HARRIS, NOEL C., and ANDREW CONSTANTI. Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AX (U.K.).
APStracts 2:0195N, 1995.
SUMMARY AND CONCLUSIONS
1. The effects of the novel bradycardic agent Zeneca ZD 7288 (4-(N-ethyl-N- phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride) were investigated on the hyper-polarization-activated cationic current ( I h ) in guinea-pig substantia nigra pars compacta (SNC) neurones 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 rest potential) elicited slow inward current relaxations with kinetic properties similar to those seen for other neuronal I h currents. 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, the I h current 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 exposure to 50 [mu]M ZD 7288, with no significant recovery observed after 1 hr washing. 4. A similar (though more rapid) block of I h was seen following application of 3-5 mM Cs + (partially reversible after 30 min wash). 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 current activation curve, a small negative shift in its position on the voltage axis and a linearization of the steady-state I- V relationship. The estimated I h current reversal potential ( E rev ) 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 -70 to -120 mV for [tau] on and -70 to -90 mV for [tau] off respectively). 7. Blockade of I h by ZD 7288 or Cs + was independent of prior I h current activation (i.e. non use-dependent). 8. Intracellular loading with ZD 7288 also abolished the 'sag' in the electrotonic voltage response and I h current 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 (t « 20 s) inward current at -100 mV holding potential. 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. 

Received 7 April 1995; accepted in final form 6 July 1995.
APS Manuscript Number J233-5.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 30 July 1995.