Properties of HERG Channels Stably Expressed in HEK 293 Cells Studied at Physiological Temperature

Abstract

We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular 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 -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, 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 physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 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 constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.

Inhibition of the current of heterologously expressed HERG potassium channels by flecainide and comparison with quinidine, propafenone and lignocaine

“…Values for t f ranged from 175 to 520 ms and for t s ranged from 1140 to 4390 ms. The mean values for both time constants that we observed under control conditions (see Figure 4) were slower than those reported by Zhou et al (1998). Using data from 16 cells, we compared mean values for each of t f and t s between control solution and FLEC-containing solution.…”

“…The I HERG tail amplitude exceeds current magnitude during voltage commands to positive voltages due to the particularly rapid voltagedependent inactivation kinetics of HERG channels (Sanguinetti et al, 1995;Trudeau et al, 1995;Smith et al, 1996;Zhou et al, 1998). Both the current during the pulse and the ensuing I HERG tail on repolarization to 740 mV were inhibited by the drug.…”

“…All experiments were performed on a cell line (Human embryonic kidney; HEK 293) stably expressing HERG, and generously donated by Professor Craig January, University of Wisconsin (Zhou et al, 1998). Cells were passaged using a non-enzymatic agent (Splittix, AutogenBioclear) and plated out onto small sterilized glass coverslips in 30 mm petri dishes containing a modi®cation of Dulbecco's Minimum Essential Medium with Glutamax-1 (DMEM; Gibco), supplemented with 10% foetal calf serum, 400 mg ml 71 gentamycin (Gibco) and 400 mg ml 71 geneticin (G418; Gibco).…”

“…At 35 ± 378C, I HERG is known to activate rapidly upon depolarization to a positive membrane potential (Hancox et al, 1998;Zhou et al, 1998). Thus, in order to determine the e ects of FLEC, I HERG was elicited by 500 ms duration test pulses to +30 mV from a holding potential of 740 mV (pulse frequency of 0.2 Hz).…”

“…Snyders & Chaudhary, 1996;Zhou et al, 1998). Some other HERG-blocking agents show a di erent pro®le, with blockade evident early during depolarization (Teschemacher et al, 1999;Walker et al, 2000;Mergenthaler et al, 2001;Witchel et al, 2002).…”