Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra

Abstract

We have characterized the native voltage-dependent K+ (Kv) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with Kv2.1 and Kv2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEKKv2.1 and HEKKv2.2). RUSMC were perfused with Hanks′ solution at 37°C and studied using the patch-clamp technique with K+-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca2+-activated K+ (BK) currents and depolarized to +40 mV for 500 ms to evoke Kv currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3–5) but were blocked by stromatoxin-1 (ScTx, IC50 ∼130 nM), consistent with the idea that the currents were carried through Kv2 channels. RNA was detected for Kv2.1, Kv2.2, and the silent subunit Kv9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both Kv2 subtypes and Kv9.3 in isolated RUSMC. HEKKv2.1 and HEKKv2.2 currents were blocked in a concentration-dependent manner by ScTx, with estimated IC50 values of ∼150 nM (Kv2.1, n = 5) and 70 nM (Kv2.2, n = 6). The mean half-maximal voltage ( V1/2) of inactivation of the USMC Kv current was −56 ± 3 mV ( n = 9). This was similar to the HEKKv2.1 current (−55 ± 3 mV, n = 13) but significantly different from the HEKKv2.2 currents (−30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that Kv2.1 channels contribute significantly to the Kv current in RUSMC.