Muscarinic receptor activation of potassium channels in rat dentate gyrus neurons

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.