MUSCARINIC RECEPTOR HYPERPOLARIZES COCHLEAR HAIR-CELLS OF CHICK BY ACTIVATING CA2+-ACTIVATED K+ CHANNELS

1. Electrical responses to extracellularly applied acetylcholine (ACh) and to intracellularly introduced substances were studied in isolated short and tall hair cells from the chick cochlear organ by a whole-cell voltage clamp technique using a patch electrode. These cells were isolated without using proteolytic enzymes. 2. Short hair cells generated a transient outward current at -50 mV in normal saline in response to puff-applied 100-mu-M-ACh, when the patch electrode was filled with a 160 mM-K+ and 100-mu-M-EGTA-based intracellular medium. The amplitude was 317.1 +/- 97.1 pA (n = 32). When ACh was applied ionophoretically, the outward current was generated with a delay of about 10 ms. 3. The amplitude of ACh-induced current was dose dependent with a K(D) of 19-mu-M and a Hill coefficient of 1.6 when measured at -50 mV. 4. The ACh (100-mu-M)-induced current was suppressed by 1-mu-M-atropine. ACh-induced current was generated in a Ca2+-free extracellular medium; however, the second ACh puff in the Ca2+-free medium generated a much reduced response. ACh-induced current was suppressed reversibly by 100-mu-M-quinine. 5. Intracellular injections of guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), inositol 1,4,5-trisphosphate (IP3) or Ca2+ (1-mu-M) via the patch pipette activated outward currents at -50 mV. 6. When the internal medium with strong Ca2+-buffering capacity (5mM-EGTA) was used, the ACh-induced current was reduced to 39.3 +/- 6.8 pA (n = 4) at -50 mV (12.3% of the response in the low-EGTA medium). 7. The reversal potential of the ACh-induced current was -85.7 +/- 4.2 mV (n = 3) in normal saline containing 5 mM-K+. The reversal potential was dependent on the extracellular K+ concentration ([K+]o) and was shifted 57 mV by a 10-fold increase in [K+]o at room temperature (20-25-degrees-C). 8. These results (points 4-7) indicate that ACh induces a K+ conductance by releasing Ca2+ intracellularly, probably by activating the pathway of muscarine receptor, G-protein and IP3. 9. Channel activities were recorded using cell-attached patch electrodes. Channel activities were rarely observed when ACh was applied to the extra-patch membrane, while robust channel activities were observed when ACh was included in the patch pipette medium. It is therefore suggested that Ca2+-activated K+ channels exist in the membrane in close vicinity to muscarinic receptor molecules and intracellular Ca2+ release sites. The single channel conductance of the ACh-induced K+ current was 83.4 +/- 4.2 pS (n = 3). 10. Tall hair cells generated much smaller currents than short hair cells, even though ACh was puff applied for a much longer period of time: 41.3 +/- 14.6 pA (n = 9) outward current was observed at -50 mV with a delay of 36.4 +/- 20.5 s (n = 9) after a 1 s puff. The reversal potential of the increased conductance was -90 mV in normal saline and was shifted 58 mV by the 10-fold increase in [K+]o.