ELECTROPHYSIOLOGICAL AND PHARMACOLOGICAL CHARACTERIZATION OF A MAMMALIAN SHAW CHANNEL EXPRESSED IN NIH 3T3 FIBROBLASTS

1. The Shaw-like voltage-activated potassium channel Kv3.1 is expresse d in neurons that generate rapid trains of action potentials. By expre ssing this channel in a mammalian cell line and by simulating its acti vation, we tested the potential role of this channel in action potenti al repolarization. 2. NIH 3T3 fibroblasts were stably transfected with Kv3.1 DNA. Currents recorded in these cells had a threshold of activa tion at approximately -10 mV, showed little inactivation, and were ver y sensitive to blockade by 4-aminopyridine and tetraethylammonium. 3. Kv3.1 currents activated rapidly at the onset of depolarizing voltage pulses. After an initial rapid phase of activation, which could be fit by an n(4) Hodgkin-Huxley model, Kv3.1 currents expressed in fibrobla sts had a second, slower phase of activation, and, in some cells, a sl ower phase of partial inactivation, both of which could be fit with mo dified n(4)p models. 4, Cell-attached single-channel recordings indica ted that the Kv3.1 channel displays two gating behaviors, a short-open -time pattern, which occurs only at the onset of depolarization, and a long-open-time pattern, which predominates during prolonged depolariz ations. 5. The amplitude of Kv3.1 currents, and the probability of cha nnel openings, was reduced by a phorbol ester activator of protein kin ase C, and the action of this agent was blocked by preincubation with the protein kinase inhibitor H7 (1-[5-isoquinolinesulfonyl]-2-methyl p iperazine). In contrast, the effects of dioctanoyl glycerol, which als o attenuated the currents, could not be completely blocked by H7, sugg esting that diacylglycerols may act on the channel by a kinase-indepen dent pathway. 6. Incorporation of a current with the kinetics and volt age dependence of Kv3.1 currents into a model cell with a sustained in ward current showed that, in contrast to other delayed-rectifier curre nts such as the Shaker-like Kv1.1 and Kv1.6 channels, the level of exp ression of Kv3.1 currents could be varied over a wide range without at tenuation of action potential height. Our results suggest that the Kv3 .1 channel may provide rapidly firing neurons with a high safety facto r for impulse propagation.