A patch‐clamp study of K+‐channel activity in bovine isolated tracheal smooth muscle cells

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Abstract
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 μm. 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 μm) 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 μm. 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 μm) 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 μm) 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.