Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels.
KChIPs (K+ channel interacting proteins) regulate the function of A-type Kv4 potassium channels by modifying channel properties and by increasing their cell surface expression. We have explored factors affecting the localisation of Kv4.2 and the targeting of KChIP1 and other NCS proteins by using GFP-variant fusion proteins expressed in HeLa cells. ECFP-Kv4.2 expressed alone was not retained in the ER but reached the Golgi complex. In cells co-expressing ECFP-Kv4.2 and KChIP1-EYFP, the two proteins were co-localised and were mainly present on the plasma membrane. When KChIP1-EYFP was expressed alone it was instead targeted to punctate structures. This was distinct from the localisation of the NCS proteins NCS-1 and hippocalcin, which were targeted to the trans-Golgi network (TGN) and plasma membrane. The membrane localisation of each NCS protein required myristoylation and minimal myristoylation motifs of hippocalcin or KChIP1 were sufficient to target fusion proteins to either TGN/plasma membrane or to punctate structures. The existence of targeting information within the N-terminal motifs was confirmed by mutagenesis of residues corresponding to three conserved basic amino acids in hippocalcin and NCS-1 at positions 3, 7 and 9. Residues at these positions determined intracellular targeting to the different organelles. Myristoylation and correct targeting of KChIP1 was required for the efficient traffic of ECFP-Kv4.2 to the plasma membrane. Expression of KChIP1(1-11)-EYFP resulted in the formation of enlarged structures that were positive for ERGIC-53 and beta-COP. ECFP-Kv4.2 was also accumulated in these structures suggesting that KChIP1(1-11)-EYFP inhibited traffic out of the ERGIC. We suggest that KChIP1 is targeted by its myristoylation motif to post-ER transport vesicles where it could interact with and regulate the traffic of Kv4 channels to the plasma membrane under the influence of localised Ca2+ signals.
The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.
Dermott W
DW
O'Callaghan
Burcu
B
Hasdemir
Mark
M
Leighton
Robert D
RD
Burgoyne
D020816:Amino Acid Motifs; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D020756:Coatomer Protein / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D051597:Hippocalcin; D006801:Humans; D007150:Immunohistochemistry; D051605:Kv Channel-Interacting Proteins; D037241:Mannose-Binding Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D015221:Potassium Channels / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051672:Shal Potassium Channels
D002135:Calcium-Binding Proteins; D020756:Coatomer Protein; C496691:HPCA protein, human; C497018:KCND2 protein, human; C496750:KCNIP1 protein, human; D051605:Kv Channel-Interacting Proteins; C084004:LMAN1 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D051672:Shal Potassium Channels; C101464:frequenin calcium sensor proteins; D051597:Hippocalcin; D002118:Calcium