Characterisation of erg K+ currents in murine vascular smooth muscle myocytes under quasi-physiological conditions

Shuk Yin M. Yeung & Iain A. Greenwood. Ion Channels and Cell Signalling Research Centre, St George’s, University of London, Cranmer Terrace, SW17 0RE.

KCNQ encoded K+ channels have been characterised extensively in neurones and cardiomyocytes but their presence in vascular myocytes remains poorly studied. To date only Ohya et al. (2002) have shown ERG1 expression in vascular myocytes (mouse portal vein or mPV). The ensuing voltage-clamp recordings resulted in the recording of inward K+ currents with distinctive ‘hooked’ kinetics when the cells were bathed in an external solution containing 140 mM K +, 4-aminopyridine and tetraethylammonium (TEA). The present study aimed to extend these studies by characterising ERG currents in mPV myocytes bathed in an external solution containing K+ at more physiological concentration and in the absence of 4-AP or TEA using three structurally dissimilar selective ERG channel inhibitors – E-4031 dofetilide and the peptide toxin BeKm-I.

Female BALB/c mice aged 6 to 8 weeks were sacrificed by cervical dislocation and single mPV cells were obtained by enzymatic dispersion. Recordings were made using the whole cell voltage-clamp techniques with an external solution of (mM): NaCl 126, KCl 5, MgCl2 1, CaCl2 0.1, glucose 11, HEPES 10, adjusted to pH 7.2 with NaOH. The internal solution contained (mM): KCl 130, MgCl2 1, ATP 2, HEPES 10, EGTA 5, adjusted to pH 7.2 with KOH. Currents were evoked from a holding potential (V H) of –60 mV. All data are the mean of n cells from at least 2 different animals ± s.e.m.

Depolarisation from -60 mV generated a gross outward current that was followed upon repolarisation to -140 mV by an inward current with ‘hooked’ kinetics that had a mean amplitude of –124.5 ± 14.1 pA (n=14) and which was enhanced by raising the external K+ to 36 mM (n=4). Repolarisation to less negative potentials slowed both the rising and declining phases of the inward current. All hooked currents were abolished irreversibly by 1μ M E-4031 (n=4) and 1μ M dofetilide (n=6). These currents were also inhibited reversibly by BeKm-1 (1-30 nM) in a manner that implied a preference for block of closed channels. The conductance-voltage (G-V) relationship for the inward current at -140mV was fitted with a Boltzmann function to give V½ (activation) of +2.7 ± 2.3 mV (k = 17.6 ± 1.4 mV, n=12-14). This was not significantly different from the voltage-dependence for the dofetilide-sensitive current (V½Dofet-sen = -1.2 ± 3.8 mV, n=6).

Use of three selective blockers of I ERG show that robust currents generated by ERG channels exist under quasi-physiological conditions in murine portal vein myocytes.

Ohya, S., Horowitz, B. & Greenwood, I.A. (2002). Am J Physiol (Cell Physiol). 283, 866-877

This work was supported by the British Heart Foundation. Dofetilide was a kind gift from Pfizer.