Patch-clamp study of isolated taste receptor cells of the frog

Abstract

Taste discs were dissected from the tongue of R. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110 mM KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was -65.2 mV and the slope resistance 150 to 750 M omega. Pulse-depolarization from a holding voltage of -80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at -25 mV and was half-maximal at -8 mV. Steady-state inactivation was half-maximal at -67 mV and complete at -50 mV. Peak Na current averaged -0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5 mM) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5 mM) and 4-amino-pyridine (1 mM) did not block, but 5 mM Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted the IK(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10 mM Ca caused a reversible 5- to 10-mV depolarization in the current-clamp mode. Quinine (0.1 mM, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5 mM in the external solution or 0.5 microM in the pipette) caused reversible depolarization (to -40 to -20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)