PROPERTIES OF HERG CHANNELS STABLY EXPRESSED IN HEK-293 CELLS STUDIEDAT PHYSIOLOGICAL TEMPERATURE

We have established stably transfected HEK 293 cell lines expressing h igh levels of functional human ether-a go-go-related gene (HERG) chann els. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological propert ies of HERO channel current at 35 degrees C. HERG-transfected cells ex pressed an mRNA band at 4.0 kb. Western blot analysis showed two prote in bands (155 and 135 kDa) slightly larger than the predicted molecula r mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -5 0 mV, maximum current was reached with depolarizing steps to -10 mV, a nd the current amplitude declined at more positive voltages, similar t o HERG channel current expressed in other heterologous systems. Curren t density at 35 degrees C, compared with 23 degrees C, was increased b y more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, in activation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I-Kr) at physiolo gical temperatures. HERO channels were highly selective for K+. When w e used an action potential clamp technique, HERG current activation be gan shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude d uring phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potentia l, and deactivation of HERO current could participate in phase 4 depol arization, HERG current was blocked by low concentrations of E-4031 (I C50 7.7 nM), a value close to that reported for I-Kr in native cardiac myocytes. Our data support the postulate that HERG encodes a major co nstituent of I-Kr and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into t he postrepolarization period.