Massive Ca-induced membrane fusion and phospholipid changes triggered by reverse Na/Ca exchange in BHK fibroblasts.
Baby hamster kidney (BHK) fibroblasts increase their cell capacitance by 25-100% within 5 s upon activating maximal Ca influx via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fluo-5N, transiently exceeds 0.2 mM with total Ca influx amounting to approximately 5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coefficient approximately 2). Capacitance can return to baseline in 1-3 min, and responses can be repeated several times. The membrane tracer, FM 4-64, is taken up during recovery and can be released at a subsequent Ca influx episode. Given recent interest in signaling lipids in membrane fusion, we used green fluorescent protein (GFP) fusions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and diacylglycerol (DAG) binding domains to analyze phospholipid changes in relation to these responses. PI(4,5)P(2) is rapidly cleaved upon activating Ca influx and recovers within 2 min. However, PI(4,5)P(2) depletion by activation of overexpressed hM1 muscarinic receptors causes only little membrane fusion, and subsequent fusion in response to Ca influx remains massive. Two results suggest that DAG may be generated from sources other than PI(4,5)P in these protocols. First, acylglycerols are generated in response to elevated Ca, even when PI(4,5)P(2) is metabolically depleted. Second, DAG-binding C1A-GFP domains, which are brought to the cell surface by exogenous ligands, translocate rapidly back to the cytoplasm in response to Ca influx. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P(2)-binding peptides, and PLD modification by butanol do not block membrane fusion. The cationic agents, FM 4-64 and heptalysine, bind profusely to the extracellular cell surface during membrane fusion. While this binding might reflect phosphatidylserine (PS) "scrambling" between monolayers, it is unaffected by a PS-binding protein, lactadherin, and by polylysine from the cytoplasmic side. Furthermore, the PS indicator, annexin-V, binds only slowly after fusion. Therefore, we suggest that the luminal surfaces of membrane vesicles that fuse to the plasmalemma may be rather anionic. In summary, our results provide no support for any regulatory or modulatory role of phospholipids in Ca-induced membrane fusion in fibroblasts.
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Alp
A
Yaradanakul
Tzu-Ming
TM
Wang
Vincenzo
V
Lariccia
Mei-Jung
MJ
Lin
Chengcheng
C
Shen
Xinran
X
Liu
Donald W
DW
Hilgemann
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D006224:Cricetinae; D025802:Electric Capacitance; D004594:Electrophysiology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005989:Glycerides / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D010712:Phosphatidic Acids / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D054803:Phospholipase C delta / Q000235:genetics / Q000378:metabolism; D010740:Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000502:physiology / Q000648:ultrastructure
D005989:Glycerides; D026901:Membrane Transport Proteins; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010743:Phospholipids; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D000266:Adenylyl Imidodiphosphate; D011493:Protein Kinase C; D010740:Phospholipases; D010749:Phosphoprotein Phosphatases; C518024:PLCD1 protein, human; D054803:Phospholipase C delta; D002118:Calcium