Ca2+ activation of hSlo K+ channel is suppressed by N-terminal GFP tag.
The human slow poke (hSlo) K+ channel was tagged with GFP (green fluorescent protein) at the N-terminus of its alpha-subunit. The fusion protein was expressed transiently in HEK293 cells; it formed functional voltage-gated channels as shown by whole cell patch-clamp measurements. However, the tag lowered the voltage dependence of gating and it suppressed the typical left-shift of gating by intracellular binding of Ca2+. The location of the GFP-tagged N-terminus was confirmed to be on the extracellular side by application of a monoclonal antibody to nonpermeabilized cells. Structural interpretations of the effects are discussed.
Meyer
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Department of Membrane and Neurophysics, Max-Planck-Institute for Biochemistry, D-82152 Martinsride/München, Germany.
Fromherz
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D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D007668:Kidney / Q000166:cytology; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008164:Luminescent Proteins / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology*; D024681:Potassium Channels, Calcium-Activated; D014162:Transfection
D017931:DNA Primers; D007202:Indicators and Reagents; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008164:Luminescent Proteins; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D049452:Green Fluorescent Proteins; D002118:Calcium