Contribution of Kv 2 . 1 channels to the delayed rectifier current in freshly 1 dispersed smooth muscle cells from rabbit urethra

47 48 We have characterized the native voltage-dependent K + (K v) current in rabbit urethral smooth 49 muscle cells (RUSMC) and compared its pharmacological and biophysical properties with 50 K v 2.1 and K v 2.2 channels cloned from the rabbit urethra and stably expressed in HEK 293 51 cells (HEK Kv2.1 and HEK Kv2.2). RUSMC were perfused with Hanks' solution at 37°C and 52 studied using the patch clamp technique with K +-rich pipette solutions. Cells were bathed in 53 100 nM penitrem A (Pen A) to block large conductance Ca 2+-activated K + (BK) currents and 54 depolarized to +40 mV for 500 ms to evoke K v currents. These were unaffected by 55 margatoxin, κ-dendrotoxin or α-dendrotoxin (100 nM, n=3-5), but were blocked by 56 stromatoxin-1 (ScTx, IC 50 ~130 nM), consistent with the idea that the currents were carried 57 through K v 2 channels. RNA was detected for K v 2.1 K v 2.2 and the silent subunit K v 9.3 in 58 urethral smooth muscle. Immunocytochemistry showed membrane staining for both K v 2 59 subtypes and K v 9.3 in isolated RUSMC. HEK Kv2.1 and HEK Kv2.2 currents were blocked in a 60 concentration dependent manner by ScTx with estimated IC 50 values of ~150 nM (K v 2.1, 61 n=5) and 70 nM (K v 2.2, n=6). The mean V 1/2 of inactivation of the USMC K v current was – 62 56±3 mV (n=9). This was similar to the HEK Kv2.1 current (–55 ± 3 mV, n=13) but 63 significantly different from the HEK Kv2.2 currents (-30 ± 3 mV, n=11). Action potentials (AP) 64 evoked from RUSMC studied under current clamp mode were unaffected by ScTx. However 65 when ScTx was applied in the presence of Pen A, the AP duration was significantly 66 prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, 67 but only after Pen A application. 68 These data suggest that K v 2.1 channels contribute significantly to the K v current in RUSMC.