Fluorescence measurements reveal stoichiometry of K+ channels formed by modulatory and delayed rectifier alpha-subunits.
Modulatory alpha-subunits, which comprise one-fourth of all voltagegated K(+) channel (Kv) alpha-subunits, do not assemble into homomeric channels, but selectively associate with delayed rectifier Kv2 subunits to form heteromeric channels of unknown stoichiometry. Their distinct expression patterns and unique functional properties have made these channels candidate molecular correlates for a broad set of native K(+) currents. Here, we combine FRET and electrophysiological measurements to determine the stoichiometry and geometry of heteromeric channels composed of the delayed rectifier Kv2.1 subunit and the modulatory Kv9.3 alpha-subunit. Kv channel alpha-subunits were fused with GFP variants, and heteromerization of different combinations of tagged and untagged alpha-subunits was studied. FRET, evaluated by acceptor photobleaching, was only observed upon formation of functional channels. Our results, obtained from two independent experimental paradigms, suggest the formation of heteromeric Kv2.1/Kv9.3 channels of fixed stoichiometry consisting of three Kv2.1 subunits and one Kv9.3 subunit. Strikingly, despite this uneven stoichiometry, we find that heteromeric Kv2.1/Kv9.3 channels maintain a pseudosymmetric arrangement of subunits around the central pore.
Kerschensteiner
Daniel
D
Department of Pharmacology, Wellcome Laboratory of Molecular Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom. d.kerschensteiner@ucl.ac.uk
Soto
Florentina
F
Stocker
Martin
M
D002460:Cell Line; D051668:Delayed Rectifier Potassium Channels; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated / Q000502:physiology*; D021122:Protein Subunits / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D051669:Shab Potassium Channels; D014162:Transfection
C485182:Cyan Fluorescent Protein; D051668:Delayed Rectifier Potassium Channels; C496988:KCNB1 protein, human; C489356:KCNS3 protein, human; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits; D011994:Recombinant Proteins; D051669:Shab Potassium Channels; D049452:Green Fluorescent Proteins