Cooperativity in Pituitary Gonadotropes

The highly cooperative nature of Ca 2 -dependent exocytosis is very important for the precise regulation of transmitter release. It is not known whether the number of binding sites on the Ca 2 sensor can be modulated or not. We have previously reported that protein kinase C (PKC) activation sensitizes the Ca 2 sensor for exocytosis in pituitary gonadotropes. To further unravel the underlying mechanism of how the Ca 2 sensor is modulated by protein phosphorylation, we have performed kinetic modeling of the exocytotic burst and investigated how the kinetic parameters of Ca 2 -triggered fusion are affected by PKC activation. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca 2 sensor (from three to two) without significantly altering the Ca 2 -binding kinetics. The reduction in the number of Ca 2 -binding steps lowers the threshold for release and up-regulates release of fusion-competent vesicles distant from Ca 2 channels. key words: exocytosis • kinetic modeling • Ca 2 dependency • fusion • protein phosphorylation I N T R O D U C T I O N Neuron and endocrine cells release neurotransmitters and hormones by highly regulated exocytosis of secretory vesicles. Calcium plays a pivotal role in triggering exocytosis. The relationship between the magnitude of Ca 2 entry and neurotransmitter release has been estimated to be approximately third order (Dodge and Rahamimoff, 1967; Augustine et al., 1985; Wu and Wu, 2001). Direct measurement of the relationship between the rate of exocytosis and the concentration of Ca 2 near release sites in a variety of neuron and endocrine cells has further supported the thirdor fourth-order cooperative nature of Ca 2 -dependent exocytosis (Heidelberger et al., 1994; Xu et al., 1998; Schneggenburger and Neher, 2000; Voets, 2000; Beutner et al., 2001). This has been taken as indirect evidence that final Ca 2 -triggered fusion requires binding of at least three Ca 2 ions to the Ca 2 sensor(s). It has been suggested that the fourth or higher order relationship between Ca 2 and exocytosis may be a unique feature of neuronal exocytosis, which is necessary for synchronizing neurotransmitter release with presynaptic Ca 2 influx. On the other hand, studies on hormone release from nonneuronal secretory cells have demonstrated a shallower dependence of exocytosis on Ca 2 of only second to third order, suggesting that variation in the number of Ca 2 -binding steps allows precise tuning of exocytosis. It has been shown that mutations in the surface residues of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex cause a decrease in the number of sequential Ca 2 -binding steps preceding exocytosis (Sorensen et al., 2002). However, it is not known whether the number of binding sites on the Ca 2 sensor can be physiologically modulated or not. Recently, we have shown that PKC activation sensitizes the Ca 2 sensor for exocytosis in pituitary gonadotropes (Zhu et al., 2002). To further uncover the underlying mechanism of how the Ca 2 sensor is modulated by phosphorylation, we have performed kinetic modeling of the experimental data and addressed the question of how the kinetic parameters for Ca 2 triggered exocytosis are influenced by PKC. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca 2 sensor from three to two, without significantly altering the Ca 2 -binding kinetics. M A T E R I A L S A N D M E T H O D S Cell Culture and Solutions Isolated gonadotropes were prepared as previously described (Zhu et al., 2002). In brief, the anterior pituitary was removed from 4to 6-wk-old male Sprague–Dawley rats that had been castrated at week 2. Cells were used 2–4 d after dispersion. Experiments were done at room temperature in a bath solution containing 140 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes, and 10 mM glucose (pH 7.4). Cells were H. Yang and H. Liu contributed equally to this work. Correspondence to Tao Xu: [email protected] Abbreviations used in this paper: DMN, DM-nitrophen; HCSP, highly Ca 2 -sensitive pool; KS, Kolmogorov-Smirnov; NP-EGTA, nitrophenylEGTA. on Jne 3, 2017 D ow nladed fom Published February 14, 2005