Functional expression of GFP-tagged Kv1.3 and Kv1.4 channels in HEK 293 cells.
Various types of voltage gated potassium channels (Kv) are responsible for setting the resting potential and shaping the membrane potential waveform in the subcellular domains of neurons. In order to visualize the expression behaviour of recombinant Kv channels, we have fused green fluorescent protein (GFP) to the N-terminal of the alpha subunits Kv1.3 and Kv1.4. In transiently transfected HEK 293 cells the GFP-Kv chimeras localize to the plasma membrane. Whole-cell voltage clamp recordings demonstrate that they form functional potassium channels. Kinetic analysis reveals that the gating kinetics of GFP-Kv1.3 are virtually indistinguishable from those displayed by its wild-type correlate. For GFP-Kv1.4 channels we find that their gating is modified in an expected manner. In response to short depolarizing voltage pulses they do not inactivate, indicating that the attached GFP interferes with the fast N-type inactivation mechanism present in wild type Kv1.4 channels. We suggest that GFP tagging of Kv channels might be a useful tool to monitor the spatiotemporal distribution of recombinant potassium channels expressed in living neurons.
Kupper
J
J
Max-Planck-Institute for Biochemistry, Department of Membrane- and Neurophysics, Martinsried, Germany. kupper@biochem.mpg.de
D002478:Cells, Cultured; D004594:Electrophysiology; D015870:Gene Expression / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000166:cytology; D051664:Kv1.3 Potassium Channel; D051665:Kv1.4 Potassium Channel; D008164:Luminescent Proteins; D008564:Membrane Potentials / Q000502:physiology; D015221:Potassium Channels / Q000235:genetics*; D024642:Potassium Channels, Voltage-Gated
D007202:Indicators and Reagents; C496973:KCNA3 protein, human; C496977:KCNA4 protein, human; D051664:Kv1.3 Potassium Channel; D051665:Kv1.4 Potassium Channel; D008164:Luminescent Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D049452:Green Fluorescent Proteins