Hamid I. Akbarali

Role of HERG-like K(+) currents in opossum esophageal circular smooth muscle.

An inwardly rectifying K+ conductance closely resembling the human ether-a-go-go-related gene (HERG) current was identified in single smooth muscle cells of opossum esophageal circular muscle. When cells were voltage clamped at 0 mV, in isotonic K+ solution (140 mM), step hyperpolarizations to -120 mV in 10-mV increments resulted in large inward currents that activated rapidly and then declined slowly (inactivated) during the test pulse in a time- and voltage- dependent fashion. The HERG K+ channel blockers E-4031 (1 μM), cisapride (1 μM), and La3+ (100 μM) strongly inhibited these currents as did millimolar concentrations of Ba2+. Immunoflourescence staining with anti-HERG antibody in single cells resulted in punctate staining at the sarcolemma. At membrane potentials near the resting membrane potential (-50 to -70 mV), this K+ conductance did not inactivate completely. In conventional microelectrode recordings, both E-4031 and cisapride depolarized tissue strips by 10 mV and also induced phasic contractions. In combination, these results provide direct experimental evidence for expression of HERG-like K+ currents in gastrointestinal smooth muscle cells and suggest that HERG plays an important role in modulating the resting membrane potential.

Monochloramine directly modulates Ca2+-activated K+ channels in rabbit colonic muscularis mucosae

BACKGROUND & AIMSnMesenteric ischemia, infection, and inflammatory bowel disease may eventuate in severe colitis, complicated by toxic megacolon with impending intestinal perforation. Monochloramine (NH(2)Cl) is a membrane-permeant oxidant generated during colitis by the large amount of ambient luminal NH(3) in the colon. Reactive oxygen metabolites can modulate smooth muscle ion channels and thereby affect colonic motility, which is markedly impaired in colitis.nnnMETHODSnEffects of NH(2)Cl on ionic currents in the innermost smooth muscle layer of the colon, the tunica muscularis mucosae, were examined using the patch clamp technique. Membrane potential in whole tissue strips was measured using high-resistance microelectrodes.nnnRESULTSnWhole cell voltage clamp experiments showed that NH(2)Cl (3-30 micromol/L) enhanced outward currents in a dose-dependent manner, increasing currents more than 8-fold at a test potential of +30 mV. Tail current analysis showed that the currents enhanced by NH(2)Cl were K(+) currents. Inhibition by tetraethylammonium and iberiotoxin suggested that these currents represented activation of large-conductance, Ca(2+)-activated K(+) channels. The membrane-impermeant oxidant taurine monochloramine, however, had no effect on whole cell currents. Single-channel studies in inside-out patches showed that NH(2)Cl increased open probability of a 257-pS channel in symmetrical (140 mmol/L) K(+). In the presence of NH(2)Cl, the steady-state voltage dependence of activation was shifted by -22 mV to the left with no change in the single-channel amplitude. The sulfhydryl alkylating agent N-ethylmaleimide prevented NH(2)Cl-induced channel activation. NH(2)Cl also hyperpolarized intact muscle strips, an effect blocked by iberiotoxin.nnnCONCLUSIONSnNH(2)Cl, at concentrations expected to be found during colitis, may contribute to smooth muscle dysfunction by a direct oxidant effect on maxi K(+) channels.

The human ether-a-go-go related gene (HERG)-like potassium channel in rabbit colonic smooth muscle cells: A potential target for the actions of cisapride

We have recently identified HERG-like K+ currents in esophageal smooth muscle where it importantly contributes to the resting membrane potential (RMP)(Akbarali et aI., Am.J.Phys. 277: C1284, 1999). Drugs that block cardiac HERG channels cause abnormalities such as long Q-T syndrome, an effect that can be induced by the gastrointestinal prokinetic agent, cisapride. The aim of this study was, therefore, to determine a) the presence and role of HERG-like currents in colonic smooth muscle and b) the potential effect of cisapride on these K+ currents. Whole cell patch-clamp experiments were carried out on freshly isolated rabbit colonic circular muscle cells. The conductance of inwardly rectifying HERG-like K+ channels in cells was enhanced by perfusing with isotonic extracellular K+ solution (140 mM K+). Under these conditions, when cells were voltageclamped at 0 mV (EK ) , step hyperpolarizations to -120 mV in 10 mV increments resulted in large inward currents that activated rapidly and then slowly inactivated in a timeand voltage-dependent fashion (Tinac .= 55 ± 5ms at -120 mY; n=4). The peak current-voltage relationship showed marked inward rectification with a reversal at EK . The amplitude of peak currents at -120 mV was -170 ± 14 pA (n=26). At potentials negative to -60 mV, prominent inactivation resulting in cross-over of the currents was observed. A U-shaped voltage-dependence for non-inactivating (window) currents occured between -80 mV and -40 mV (peak amplitude of -80 ± 4 pA; n=3) suggesting contribution of the HERG-like channels to the resting potential of the smooth muscle cells. These K+ currents were completely abolished by the selective HERG channel blocker, E-4031 (I /LM; n= 10). Cisapride also dose-dependently blocked (0.001 10 /LM; n=24) the HERG-like K+ current with an ICso of I/LM. E-4031 and cisapride-sensitive currents demonstrated strong inward rectification. In further studies, the RMP of whole muscle strips was measured using high resistance microelectrodes. Cisapride (I/LM) and E-4031 (3/LM), in the presence of atropine (I/LM) and nifedipine (I/LM), depolarized the RMP from -47 ± 2 mV to -34 ± 4 mV and from -54mV ± 3mV to -37 ± 3 mY, respectively (n=3). These studies demonstrate a) that HERG-like currents in rabbit colonic smooth muscle are involved in maintaining resting potential and b) these gastrointestinal smooth muscle K+ channels, like in the heart, are potential targets for the prokinetic agent, cisapride. Supported by NIH(DK46367)