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 expressed in neurons that generate rapid trains of action potentials. By expressing this channel in a mammalian cell line and by simulating its activation, we tested the potential role of this channel in action potential repolarization. 2. NIH 3T3 fibroblasts were stably transfected with Kv3.1 DNA. Currents recorded in these cells had a threshold of activation at approximately -10 mV, showed little inactivation, and were very 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 n4 Hodgkin-Huxley model, Kv3.1 currents expressed in fibroblasts had a second, slower phase of activation, and, in some cells, a slower phase of partial inactivation, both of which could be fit with modified n4p models. 4. Cell-attached single-channel recordings indicated 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 depolarizations. 5. The amplitude of Kv3.1 currents, and the probability of channel openings, was reduced by a phorbol ester activator of protein kinase C, and the action of this agent was blocked by preincubation with the protein kinase inhibitor H7 (1-[5-isoquinolinesulfonyl]-2-methyl piperazine). In contrast, the effects of dioctanoyl glycerol, which also attenuated the currents, could not be completely blocked by H7, suggesting that diacylglycerols may act on the channel by a kinase-independent pathway. 6. Incorporation of a current with the kinetics and voltage dependence of Kv3.1 currents into a model cell with a sustained inward current showed that, in contrast to other delayed-rectifier currents such as the Shaker-like Kv1.1 and Kv1.6 channels, the level of expression of Kv3.1 currents could be varied over a wide range without attenuation of action potential height. Our results suggest that the Kv3.1 channel may provide rapidly firing neurons with a high safety factor for impulse propagation.
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001711:Biotransformation / Q000187:drug effects; D002478:Cells, Cultured; D003198:Computer Simulation; D004594:Electrophysiology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005822:Genetic Vectors; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008959:Models, Neurological; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D014162:Transfection
D015221:Potassium Channels; D011493:Protein Kinase C