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Journal of Neurophysiology, Vol 74, Issue 6 2366-2378, Copyright © 1995 by APS
ARTICLES |
N. C. Harris and A. Constanti
Department of Pharmacology, School of Pharmacy, London, United Kingdom.
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 (Ih) 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 Ihs. 3. ZD 7288 (10-100 microM) produced a consistent abolition of the electrotonic potential sag with no effect on membrane potential or spike properties. Under voltage clamp, Ih amplitude was clearly reduced in a time- and concentration-dependent manner (apparent half-maximum blocking concentration = 2 microM); full block of Ih was typically achieved after 10-15 min of exposure to 50 microM ZD 7288, with no significant recovery observed after 1 h of washing. 4. A similar (although more rapid) block of Ih was seen after application of 3-5 mM Cs+ (partially reversible after 30 min of washing). 5. Partial block of Ih by 10 microM ZD 7288 was accompanied by a reduction in the maximum amplitude of the Ih 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 Ih reversal potential, however, remained unaffected. 6. In 10 microM ZD 7288, the time course of Ih 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 Ih by ZD 7288 or Cs+ was independent of prior Ih activation (i.e., non-use dependent). 8. Intracellular loading with ZD 7288 also abolished the sag in the electrotonic voltage response and Ih relaxations, suggesting an intracellular site of action. By contrast, intracellular Cs+ had no effect on Ih properties. 9. Block of Ih by ZD 7288 (but not Cs+) was relieved by prolonged cell hyperpolarization, manifested as a slowly developing (half-time approximately 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 Ih 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 Ih currents in controlling neuronal function.
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