Protein kinase C-dependent enhancement of activity of rat brain NCKX2 heterologously expressed in HEK293 cells.
Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.
Lee
Ju-Young
JY
National Research Laboratory for Cell Physiology, Department of Physiology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Ku, Seoul 110-799, South Korea.
Visser
Frank
F
Lee
Jae Sung
JS
Lee
Kyu-Hee
KH
Soh
Jae-Won
JW
Ho
Won-Kyung
WK
Lytton
Jonathan
J
Lee
Suk-Ho
SH
D000818:Animals; D017920:Antiporters / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004594:Electrophysiology; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000378:metabolism / Q000502:physiology*
D017920:Antiporters; C470570:SLC24A4 protein, human; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D011493:Protein Kinase C; D002118:Calcium