Massive Ca-induced Membrane Fusion and Phospholipid Changes Triggered by Reverse Na/Ca Exchange in BHK Fibroblasts

Abstract

Baby hamster kidney (BHK) fi broblasts increase their cell capacitance by 25 -100% within 5 s upon activating maximal Ca infl ux via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fl uo-5N, transiently exceeds 0.2 mM with total Ca infl ux amounting to ‫ف‬ 5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coeffi cient ≈ 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 infl ux episode. Given recent interest in signaling lipids in membrane fusion, we used green fl uorescent 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 infl ux 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 infl ux 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 infl ux. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P 2 -binding peptides, and PLD modifi cation 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 refl ect 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 fi broblasts.on May 9, 2018 jgp.rupress.org Downloaded from

PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine

“…Recent studies with Baby Hamster Kidney cells stably expressing NCX1 (BHK-NCX1) demonstrated that a repeated activation of reverse NCX1 produces a long-term inactivation or rundown of the exchanger activity (Lee and Kang, 2007;Yaradanakul et al, 2008), and we suggested that, at least in part, the Ca 2+ entered the cells through the reverse exchanger produces rundown (Lee and Kang, 2007). Recently, Hilgemann and colleagues (2008) ]i) ranges, and the contributions of phospholipases and the cleavage of PIP2 are independent of the fusion.…”

“…Second, a question of whether rundown of NCX1 is due to internalization of NCX1 proteins should be proved (Egger et al, 2005;Shen et al, 2007). This possibility, however, might be excluded because a recent work on the capacitance measurement along with NCX1 current recording clearly demonstrated that a strong membrane fusion, but not the membrane fission, occurs when cytosolic Ca 2+ is increased by reverse NCX1 (Yaradanakul et al, 2008). Since a long-lasting, irreversible rundown of NCX1 current was accompanied with massive fusion, it is unclear whether the cellular mechanisms of NCX rundown are independent of endocytosis.…”

“…Recently, Hilgemann and colleagues (2008) ]i) ranges, and the contributions of phospholipases and the cleavage of PIP2 are independent of the fusion. They also demonstrated a long-lasting, irreversible rundown of NCX1 current in response to a repeated Ca 2+ influx, albeit the mechanisms are not clear (Yaradanakul et al, 2008). Therefore, cellular mechanisms that cause the Ca 2+ -induced rundown of NCX1 are yet to be defined.…”

Massive surface membrane expansion without involvement of classical exocytic mechanisms

“…Both electrical and optical methods have revealed that diverse cell types increase their PM area substantially during large elevations of cytoplasmic Ca 2+ (Coorssen et al, 1996;Ninomiya et al, 1996;Kasai et al, 1999;Togo et al, 2003;Kreft et al, 2004;Bretscher, 2008;Cohen et al, 2008;Yaradanakul et al, 2008). PM expansion can double PM area within a few seconds in some cell types Yaradanakul et al, 2008;Bricogne, XXXX). To what extent the different PM responses occur by a single molecular mechanism remains unclear.…”

“…Ca 2+ -activated PM expansion in BHK fibroblasts is not inhibited by tetanus toxins and does not appear to use synaptotagmins as Ca 2+ sensors . In contrast to the usual function of synaptotagmins (Sudhof, 2012), PM expansion in fibroblasts has a nearly linear dependence on cytoplasmic Ca 2+ (Yaradanakul et al, 2008). As described in a companion article (Bricogne, XXXX) , PM expansion in Jurkat T-cells and HEK293 cells can be initiated by the transmembrane protein, TMEM16F (Bricogne, XXXX).…”