A PATCH-CLAMP STUDY OF K+-CHANNEL ACTIVITY IN BOVINE ISOLATED TRACHEAL SMOOTH-MUSCLE CELLS

1 Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K+-channels in these cells were studied by the patch-clamp recording technique. 2 Recordings were made from inside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10-mu-M. Large unitary currents of both Ca2+-dependent and -independent types were observed. Measured between +20 and +40 mV, the slope conductances of the channels carrying these currents were 249 +/- 18 pS and 268 +/- 14 pS respectively. 3 Lowering [K+] on the cytosolic side of the patches from 140 to 6 mM, shifted the reversal potentials of the two types of unitary current from approximately zero to >> +40 mV, suggesting that both currents were carried by K+-channels. 4 The Ca2+-dependent and -independent K+-channels detected in inside-out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1-10 mM; Cs+, 10 mM; Ba2+, 1-10 mM; quinidine, 100-mu-M) applied to the cytosolic surface of the patches. 5 Recordings were made from outside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1-mu-M. Ca2+-dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 +/- 5 pS. 6 Activity of the Ca2+-dependent K+-channel detected in outside-out patches could be inhibited by application of TEA (1 mM), Cs+ (10 mM), Ba2+ (10 mM) or quinidine (100-mu-M) to the external surface of the patch. 4-Aminopyridine (4-AP; 1 mM) was ineffective as an inhibitor. 7 The activity of the Ca2+-dependent K+-channel recorded from outside-out patches was reversibly inhibited by charybdotoxin (100 nM). 8 When whole-cell recording was performed, the application of a depolarizing voltage ramp evoked outward current which was dependent on the [Ca2+] in the recording pipette and which could be reversibly inhibited by charybdotoxin (50 nM-1-mu-M) applied to the external surface of the cell. 9 We conclude that bovine trachealis cells are richly endowed with charybdotoxin-sensitive, large conductance, Ca2+-dependent K+-channels. These channels carry most of the outward current evoked by a depolarizing ramp and could play a major role in determining the outward rectifying properties of the trachealis cells. The role of the large Ca2+-independent K+-channels remains unclear.