MUSCARINIC RECEPTOR ACTIVATION OF POTASSIUM CHANNELS IN RAT DENTATE GYRUS NEURONS

1. The effects of acetylcholine (ACh) on granule cells freshly dissoci ated from rat dentate gyrus (DG) were studied using the nystatin perfo rated patch technique. This method allowed us to study ACh-induced cur rents (I(ACh)) under voltage clamp without ''run-down'' of the ACh res ponse. 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 increas ing ACh concentration. The half-maximal response and the Hill coeffici ent determined from the relation between ACh concentration and respons e were 4.98 x 10(-7) M and 1.70, respectively. 3. The reversal potenti al 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 resp onse. 5. Muscarinic antagonists reversibly suppressed the I(ACh)(10(-5 ) M) in a concentration-dependent manner, where the values of half-inh ibition 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-beta S suppressed the I(ACh) greatly. The I(ACh) persisted in the neurons p retreated with an external solution containing pertussis toxin (IAP) f or 18 h. 7. In the neurons perfused with Ca2+-free external solution c ontaining 2 mM ethylene glycol-O,O'-bis (beta-aminoethyl ether)-N,N,N' ,N'-tetraacetic acid and 10 mM Mg2+, the first application of ACh indu ced the I(ACh) with an amplitude similar to that in the standard solut ion. However, the second application had no effect. Intracellular perf usion 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 membr ane in the conventional whole-cell mode. 8. Pretreatment with Li+ (10( -4) M) enhanced the I(ACh) amplitude at low ACh concentration. Each in tracellular perfusion of heparin and inositol trisphosphate (IP3) supp ressed the I(ACh). 9. Chlorpromazine (IC50; 4.10 x 10(-7) M) and -(6-a minohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride(IC50; 1.09 X 10(-7) M) reversibly suppressed the I(ACh) in a concentration-depen dent manner. 10. Modulators for protein kinase C such as 1-(5-isoquino linylsulfonyl)-2-methylpiperazine dihydrochloride, staurosporine, and phorbol ester, or for A kinase, such as forskolin, isobutylmethylxytha nthine, and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydro chloride, had no effect on the I(ACh). 11. It was concluded that musca rinic receptors stimulate K+ selective channels in neurons from DG. Th e pathway coupling receptor to K+ channels involves IAP-insensitive G proteins and might involve IP3-stimulated release of intracellular.