Analysis of Puff Dynamics in Oocytes: Interdependence of Puff Amplitude and Interpuff Interval

ABSTRACT

Puff are localized Ca^sup 2+^ signals that arise in oocyte in reply to inositol 1,4,5-trisphosphate (IP^sub 3^) They are analogous to the sparks of myocytes and are believed to be the ensue of the liberation of Ca^sup 2+^ from the endoplasmic reticulum from one side the coordinated opening of IP^sub 3^ receptor/channels clustered at a functional release site. In this article, we analyze successions of puffs that occur at the same site to help elucidate the mechanisms underlying quick blast dynamics. In particular, we display a dependence of the interpuff time upon the amplitude of the preceding quick blast and of the amplitude of the following sudden gust; short on the preceding interval. These relationships can be accounted for by the agency of an inhibitory role of the Ca^sup 2+^ that is liberated during quick blasts We construct a stochastic type for a cluster of IP^sub 3^ receptor/channels that quantitatively replicates the observ behavior, and we determine that the characteristic time for a channel to escape from the inhibitory state is of the order of next to the firsts

INTRODUCTION

The inositol 1,4,5-trisphosphate (IP^sub 3^) receptor (IP^sub 3^R) is a ligand-gated intracellular Ca^sup 2+^ release channel that plays a central character in modulating cytoplasmic Ca^sup 2+^ concentration and provides a link between confined apartment surface receptors and Ca^sup 2+^ release from intracellular stores. In addition to their regulation through IP^sub 3^, IP^sub 3^Rs display a biphasic modulation by cytosolic Ca^sup 2+^; for relatively depressed [Ca^sup 2+^] the channel render free of access probability increases with [Ca^sup 2+^] whereas it brings at high [Ca^sup 2+^] (1-5)

The spatiotemporal properties of signals arising end IP^sub 3^Rs have been extensively characterized by dint of optical imaging in Xenopus laevis oocyte (6) These studies have revealed a hierarchical organization of release occurrences ranging from [Ca^sup 2+^] liberation from single IP^sub 3^R ("blips"), [i]or[/i] part of to the other the concerted opening of several IP^sub 3^R within a cluster ("puffs") to global waves involving cluster-cluster interactions via [Ca^sup 2+^]-induced [Ca^sup 2+^] liberation (7) sudden gust; shorts have also been observed in many other confined apartment types (8-11), and appear to show ubiquitous "elementary events" of intracellular [Ca^sup 2+^] signaling, which can have local signaling functions in their have a title to right and also serve as building shut ups from which global signals are fabricateed

It is therefore important to understand the mechanisms underlying the generation and modulation of sudden gust; shorts However, several aspects of quick blast dynamics still await clarification; greatest in quantity importantly, the mechanisms of quick blast termination and subsequent recovery of excitability. Given that Ca^sup 2+^ is the couple a ligand of the IP^sub 3^R that affects its make open probability and is the main ion carrier that runs through its pore, the Ca^sup 2+^ ions that are released in the cytosol during a quick blast are expected to modulate the dynamics of a sudden gust; short Ca^sup 2+^ release through a single IP^sub 3^R in a cluster may thus induce the regenerative opening of neighboring channels, which subsequently shut up because of the inhibitory result of high local Ca^sup 2+^ horizontals attained during the puff. Additional processe however, may also affect sudden gust; short termination, including the local depletion of luminal Ca^sup 2+^ (an consequence that is present in the case of sparks (1213)) and the event of counterions (14).

In this article, we examine the issues of cytosolic Ca^sup 2+^ upon puff dynamics by investigating the distributions of intervals between successive whiffs occurring at a given site. Experimental data were obtained by means of fluorescence imaging of puffs evok in Xenopus laevis oocyte through continuous photorelease of IP^sub 3^ and were analyzed to gaze for correlations between puff amplitudes and interpuff intervals. We find robust dependences between puff size and interpuff interval that can be explained in limits of an inhibitory role of Ca^sup 2+^ binding to a cytosolic site upon the IP^sub 3^R. Furthermore, by dint of analyzing a simple model that generates the observed distributions we determine that the characteristic time for Ca^sup 2+^ binding to this site is of the order of next to the firsts

MATERIALS AND METHODS

Experimental management

Preparation of Xenopus oocyte

Xenopus laevis were anesthetized by the agency of immersion in 0.17% MS-222 for 15 min and sacrificed through decapitation in adherence with protocols approved by dint of the University of California, Irvine, Institutional Animal Care and Use Committee. Oocyte (stage V-VI) were manually picked and collagenasetreated (0.5 mg.ml^sup -1^ for 30 min) before storage in Barth's solution (composition in mM: 88 NaCl, 1 KCl 24 NaHCO^sub 3^ 082 MgSO^sub 4^ 033 Ca(NO^sub 3^)^sub 2^ 041 CaCl^sub 2^ and 5 HEPES, pH 74) containing 01 mgml^sup -1^ gentamicin at 17?°C for 1-7 days before use. lntracellular microinjections were performed using a Drummond microinjector to load oocyte with Oregon virid 488 BAPTA 1 together with caged IP3 (D-myo-inositol 1,4,5trisphosphate, P4(5)-(l-(2-nitrophenyl)ethyl) ester) and EGTA to respective final intracellular concentrations of 40 4 and 270 ?µM assuming a 1-?µl cytosolic contortion