1. The effects of acetylcholine (ACh) on granule cells freshly dissociated from rat dentate gyrus (DG) were studied using the nystatin perforated patch technique. This method allowed us to study ACh-induced currents (I(ACh)) under voltage clamp without 'run-down' of the ACh response. In some experiments, we used the conventional whole-cell method for intracellular application of drugs not permeable to cell membrane. 2. At a holding potential of -40 mV, ACh induced an outward current. The amplitude of I(ACh) increased in a sigmoidal fashion with increasing ACh concentration. The half- maximal response and the Hill coefficient determined from the relation between ACh concentration and response were 4.98 x 10-7 M and 1.70, respectively. 3. The reversal potential of I(ACh) was close to the K+ equilibrium potential. The I(ACh) was accompanied by an enhancement of the K+ current. 4. Muscarine and McN-A-343 mimicked the ACh response, whereas oxotremorine induced no response. 5. Muscarinic antagonists reversibly suppressed the I(ACh) (10-5 M) in a concentration-dependent manner, where the values of half-inhibition concentration (IC50) were 1.03 x 10-6 M for pirenzepine and 2.21 x 10-5 M for AF-DX-116. 6. Intracellular perfusion with GDP-βS suppressed the I(ACh) greatly. The I(ACh) persisted in the neurons pretreated with an external solution containing pertussis toxin (IAP) for 18 h. 7. In the neurons perfused with Ca2+-free external solution containing 2 mM ethylene glycol-O,O'-bis(β-aminoethyl ether)-N,N,N',N'- tetraacetic acid and 10 mM Mg2+, the first application of ACh induced the I(ACh) with an amplitude similar to that in the standard solution. However, the second application had no effect. Intracellular perfusion with 2 mM 1,2- bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked the I(ACh) completely within 3 min after rupture of membrane in the conventional whole- cell mode. 8. Pretreatment with Li+ (10-4 M) enhanced the I(ACh) amplitude at low ACh concentration. Each intracellular perfusion of heparin and inositol trisphosphate (IP3) suppressed the I(ACh). 9. Chlorpromazine (IC50; 4.10 x 10-7 M) and N-(6-amino-hexyl)-5-chloro-1-naphthalene- sulfonamide hydrochloride (IC50; 1.09 x 10-7 M) reversibly suppressed the I(ACh) in a concentration-dependent manner. 10. Modulators for protein kinase C such as 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride, staurosporine, and phorbol ester, or for A kinase, such as forskolin, isobutyl-methylxythanthine, and N-[2-(methylamino)ethyl]-5- isoquinolinesulfonamide dihydrochloride, had no effect on the I(ACh). 11. It was concluded that muscarinic receptors stimulate K+ selective channels in neurons from DG. The pathway coupling receptor to K+ channels involves IAP- insensitive G proteins and might involve IP3-stimulated release of intracellular.
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