The role of the C-terminus for functional heteromerization of the plant channel KDC1.
Voltage-gated potassium channels are formed by the assembly of four identical (homotetramer) or different (heterotetramer) subunits. Tetramerization of plant potassium channels involves the C-terminus of the protein. We investigated the role of the C-terminus of KDC1, a Shaker-like inward-rectifying K(+) channel that does not form functional homomeric channels, but participates in the formation of heteromeric complexes with other potassium alpha-subunits when expressed in Xenopus oocytes. The interaction of KDC1 with KAT1 was investigated using the yeast two-hybrid system, fluorescence and electrophysiological studies. We found that the KDC1-EGFP fusion protein is not targeted to the plasma membrane of Xenopus oocytes unless it is coexpressed with KAT1. Deletion mutants revealed that the KDC1 C-terminus is involved in heteromerization. Two domains of the C-terminus, the region downstream the putative cyclic nucleotide binding domain and the distal part of the C-terminus called K(HA) domain, contributed to a different extent to channel assembly. Whereas the first interacting region of the C-terminus was necessary for channel heteromerization, the removal of the distal K(HA) domain decreased but did not abolish the formation of heteromeric complexes. Similar results were obtained when coexpressing KDC1 with the KAT1-homolog KDC2 from carrots, thus indicating the physiological significance of the KAT1/KDC1 characterization. Electrophysiological experiments showed furthermore that the heteromerization capacity of KDC1 was negatively influenced by the presence of the enhanced green fluorescence protein fusion.
Naso
Alessia
A
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa, Italy.
Dreyer
Ingo
I
Pedemonte
Laura
L
Testa
Ilaria
I
Gomez-Porras
Judith Lucia
JL
Usai
Cesare
C
Mueller-Rueber
Bernd
B
Diaspro
Alberto
A
Gambale
Franco
F
Picco
Cristiana
C
D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics; D018552:Daucus carota / Q000737:chemistry*; D055724:Electrophysiological Phenomena; D005260:Female; D005453:Fluorescence; D009865:Oocytes / Q000166:cytology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques
D029681:Arabidopsis Proteins; C501210:KAT1 protein, Arabidopsis; C419388:KDC1 protein, Daucus carota; D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying