Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis

Synexin was isolated from bovine liver and found to aggregate adrenal chromaffin granules in the same Ca2+-dependent manner as previously described for adrenal synexin. The chromaffin granule aggregating activity of liver synexin was blocked in vitro by the addition of an antibody prepared to the 47,000 molecular weight band extracted from an SDS gel of an adrenal medullary synexin preparation. Chromaffin granules aggregated by synexin fused when exposed to cis-unsaturated fatty acids at concentrations comparable to those released from phospholipids by stimulated secretory cells. The synexin-induced aggregation reaction was blocked by Erythrosin B, a common food coloring, and by the phenothiazine antipsychotic trifluoperazine and promethazine. The aggregation and fusion of chromaffin granules thus appears to be a useful model system for studying synexin from diverse tissues and for testing pharmacologically or toxicologically active substances for effects on secretory systems.     

Stimulus-Secretion Coupling in Isolated Adrenal Chromaffin Cells: Calcium Channel Activation and Possible Role of Cytoskeletal Elements

Abstract: The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus-secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel-blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh- and K-evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage-dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K-evoked secretion to levels comparable to that of ACh but did not induce a vinblastine block of K-evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.     

A K+-selective,three-state channel from fragmented sarcoplasmic reticulum of frog leg muscle

Summary Sarcoplasmic reticulum (SR) vesicles from frog leg muscle were fused with a planar phospholipid bilayer by a method described previously for rabbit SR. As a result of the fusion, K⁺-selective conduction channels are inserted into the bilayer. Unlike the two-state rabbit channel, the frog channel displays three states: a nonconducting (closed) state and two conducting states and . In 0.1m K⁺ the single-channel conductances are 50 and 150 pS for and , respectively. The probabilities of appearearance of the three states are voltage-dependent, and transitions between the closed and states proceed through the state. Both open states follow a quantitatively identical selectivity sequence in channel conductance: K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺>Cs⁺. Both open states are blocked by Cs⁺ asymmetrically in a voltage-dependent manner. The zero-voltage dissociation constant for blocking is the same for both open states, but the voltage-dependences of the Cs⁺ block for the two states differ in a way suggesting that the Cs⁺ blocking site is located more deeply inside the membrane in the than in the state.     

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum

Summary The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca²⁺ transport. The absorbance and fluorescence responses of 3,3-diethyl-2,2-thiadicarbocyanine, 3,3-dimethyl-2,2-indodicarbocyanine and oxonol VI during ATP-dependent Ca²⁺ transport are influenced by the effect of accumulated Ca²⁺ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca²⁺ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca²⁺, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulumin vivo andin vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca²⁺ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca²⁺ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca²⁺ translocation. The potential generated during Ca²⁺ transport is rapidly dissipated by passive ion fluxes across the membrane.     

Structure and Disorder in an Unfolded State under Nondenaturing Conditions from Ensemble Models Consistent with a Large Number of Experimental Restraints

Obtaining detailed structural models of disordered states of proteins under nondenaturing conditions is important for a better understanding of both functional intrinsically disordered proteins and unfolded states of folded proteins. Extensive experimental characterization of the drk N-terminal SH3 domain unfolded state has shown that, although it appears to be highly disordered, it possesses significant nonrandom secondary and tertiary structure. In our previous attempts to generate structural models of the unfolded state using the program ENSEMBLE, we were limited by insufficient experimental restraints and conformational sampling. In this study, we have vastly expanded our experimental restraint set to include ¹H-¹⁵N residual dipolar couplings, small-angle X-ray scattering measurements, nitroxide paramagnetic relaxation enhancements, O₂-induced ¹³C paramagnetic shifts, hydrogen-exchange protection factors, and ¹⁵N R₂ data, in addition to the previously used nuclear Overhauser effects, amino terminal Cu²⁺-Ni²⁺ binding paramagnetic relaxation enhancements, J-couplings, chemical shifts, hydrodynamic radius, and solvent accessibility restraints. We have also implemented a new ensemble calculation methodology that uses iterative conformational sampling and seeks to calculate the simplest possible ensemble models. As a result, we can now generate ensembles that are consistent with much larger experimental data sets than was previously possible. Although highly heterogeneous and having broad molecular size distributions, the calculated drk N-terminal SH3 domain unfolded-state ensembles have very different properties than expected for random or statistical coils and possess significant nonnative α-helical structure and both native-like and nonnative tertiary structure.