The inhibitory effect of the melatonin receptor antagonist luzindole on voltage-activated transient outward K current (I) was K(A) investigated in cultured rat cerebellar granule cells using the whole cell voltage-clamp technique. At the concentration of 1 mM to 1 mM,luzindole reversibly inhibited I in a concentration-dependent manner. In addition to reducing the current amplitude of I,luzindole K(A) K(A) accelerated the fast inactivation of I channels and shifted the curves of voltage-dependent steady-state activation and inactivation of K(A) I by 16.6 mV and 27.0 mV, respectively. The inhibitory effect of luzindole was neither use-dependent nor voltage-dependent, K(A) suggesting that the binding affinity of luzindole to I channels is state-dependent. Including luzindole in the pipette solution, or K(A) extracellular application of 4 P-PDOT, an antagonist of melatonin receptors, did not change the luzindole-induced inhibitory effect on the I current, indicating that luzindole exerts its channel blocking inhibitory action at the extracellular mouth of the channel, and that the K(A)effect is not due to action of the melatonin receptors. Our data are the first demonstration that luzindole is able to block transient outward 1 K channels in rat cerebellar granule cells in a state-dependent manner, likely associated with extracellular interaction of the drug with the I inactivation gate.

Swelling-activated Cl? currents, I(Cl,swell), were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique. Genistein, an inhibitor of protein tyrosine kinase (PTK), suppressed I(Cl,swell). Under isosmotic condition phorbol 12-myristate 13-acetate (PMA), an activator of PKC, elicited the Cl? current similar to that in hyposmotic solution, whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells. Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition. When the cells were treated with cytochalasin B (CB) or cytochalasin D (CD) under isosmotic condition the disruption of the F-actin integrity was observed, and I(Cl,swell) was not elicited. With combination treatment of CB with PMA, hyposmotic solution could not elicited I(Cl,swell). The results suggested that the role of PTK, probably receptor tyrosine kinase, for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin. Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl? channels. These results demonstrate clearly that PTK, PKC and F-actin are important factors for regulation of I(Cl,swell), in embryonic chick heart cells as compared with often controversial results reported in different cell types.

The effects of adenosine on the voltage-sensitive delayed-rectifier Kq I. currents and hyperpolarization-activated cationic inward K current Ih. were studied in cultured frog melanotrophs using the whole-cell configuration of the patch-clamp technique. The A1 receptor agonist R-N6-phenylisopropyl-adenosine R-PIA; 50 mM. reversibly increased I. Perfusion of dibutyryl-cAMP 1 mM. in the external K solution did not modify the R-PIA-induced enhancement of IK. Pretreatment of melanotrophs with pertussis toxin 1 mgrml; 12 h.totally abolished the R-PIA-evoked response. Application of hyperpolarizing voltage pulses from y60 to y120 mV to melanotrophs induced a two-component inward current corresponding to an I-like conductance. This conductance was characterized by a high Kq hselectivity and a low Naq permeability and was resistant to etrodotoxin 1 mM.. R-PIA had no effect on I. The present study hdemonstrates that in frog melanotrophs adenosine inhibits the electrical activity by activating IK through an A1 receptor subtype coupled to a pertussis toxin-sensitive pathway independent of the cAMPrPKA system. This study also demonstrates the existence of a Ih conductance in frog melanotrophs which is not modulated by A1 receptors. q1998 Elsevier Science B.V. All rights reserved.

1. Transient outward current was recorded in cultured frog melanotrophs with the whole-cell configuration of the patch-clamp technique. The ionic dependence, kinetics and pharmacological properties of the current were studied. The effects of the A1 adenosine receptor agonist R-N6 enylisopropyl-adenosine (R-PIA) on this current were also investigated. 2. In tetrodotoxin-and cobalt-containing solution, depolarization from -120 mV elicited both transient and delayed outward currents. Pulses from -60 mV activated only a sustained late current. 3. 4-Aminopyridine (4 mM) reduced the transient outward current much more than the delayed outward current. In contrast, tetraethylammonium (10-20 mM) selectively reduced the delayed current. 4. Tail current measurements showed a positive shift in the reversal potential when external K+ concentration was increased, indicating that K+ was the predominant charge carrier. 5. Steady-state inactivation was complete at potentials positive to -10 mV and removed by hyperpolarization. 6. Inactivation of the transient current was slowed and accelerated in oxidizing and reducing conditions, respectively, confirming the involvement of an inactivating 'ball and chain'peptide. 7. R-PIA increased the transient current. The steady-state inactivation curve was shifted towards more positive potentials without changing the activation kinetics. Pretreatment with pertussis toxin (1 jug ml-') blocked the response to R-PIA. 8. It is concluded that frog melanotrophs possess an A-type current that is likely to play an important role in excitability. This current, which is directly modulated by A, adenosine receptors through a Gi/Go protein, appears to be responsible for the inhibitory effects of adenosine on electrical activity.

The effects of 4-aminopyridine (4-AP), a specific blocker of outward K current, on voltage-activated transient outward K current 1(I) and inward Na current (I) were investigated on cultured rat cerebellar granule cells using the whole cell voltage-clamp K(A) Na 1technique. At the concentration of 1–5 mM, 4-AP inhibited both I and I. It reduced the amplitude of peak Na current without K(A) Na significant alteration of the steady-state activation and inactivation properties. The inhibitory effect was not enhanced by repeated 1depolarizing pulses (0.5 or 0.1 Hz), suggesting that the binding affinity of 4-AP on Na channels is state-independent. In contrast, the 1 effect of 4-AP on Na channels appeared to be voltage-dependent, the weaker inhibition occurred at more depolarization. Moreover, 4-AP 1 slowed both the activation and inactivation kinetics of Na current. These effects were similar to those induced by a-scorpion toxin and 1 sea anemone toxins on Na channels in other cell model. Our data demonstrate for the first time that 4-AP is able to block not only 11 1 A-type K channels, but also Na channels in rat cerebellar granule cells. It is concluded that the inhibition exerted by 4-AP on Na current likely differs from that provoked by local anesthetics. The possibility that the binding site of neurotoxin receptor 3 may be involved is discussed.