Calcium channels are well known targets for inhibition by G protein-coupled receptors, and multiple forms of inhibition have been described. Here we report a novel mechanism for G protein-mediated modulation of neuronal voltage-dependent calcium channels that involves the destabilization and subsequent removal of calcium channels from the plasma membrane. Imaging experiments in living sensory neurons show that, within seconds of receptor activation, calcium channels are cleared from the membrane and sequestered in clathrin-coated vesicles. Disruption of the L1-CAMankyrin B complex with the calcium channel mimics transmitterinduced trafficking of the channels, reduces calcium influx, and decreases exocytosis. Our results suggest that G protein-induced removal of plasma membrane calcium channels is a consequence of disrupting channel-cytoskeleton interactions and might represent a novel mechanism of presynaptic inhibition.Dunlap and Fischbach (1) have suggested that transmitter-mediated shortening of the duration of the action potential could be due to a decrease in calcium conductance or a decrease in the number of functional channels in the membrane. Because of the importance of such a mechanism for the regulation of synaptic transmission, much attention has been placed to the mechanisms of receptor-mediated modulation of voltage-gated calcium channels. Inhibition of Ca 2ϩ channels can be voltage-dependent and is mediated by direct interaction of G protein ␤␥ subunits with the ␣1 pore-forming subunit of the channel (2, 3). In addition, phosphorylation by kinases such as protein kinase C and tyrosine kinases has been shown to inhibit Ca 2ϩ channels (4). Subsequent work has established that G protein-dependent inhibition of calcium current is in part a result of a decrease in the open probability of the channel, reducing current density (5-7). The idea that changes in channel density could underlie calcium channel modulation has not been tested.Activity-and receptor-dependent trafficking of ionotropic receptors has been widely studied in the post-synaptic density (8, 9). Such studies have not been extended to proteins in the presynaptic active zones. In this study we have found that activation of G protein-coupled receptors induces destabilization and subsequent removal of calcium channels from the plasma membrane. Transmitter-induced trafficking of calcium channels is a consequence of disrupting the interaction of the channel with L1-CAM and ankyrin B and might represent a novel mechanism of presynaptic inhibition.

Exploration for unconventional reservoirs has begun in various countries in the Middle East. Widely recognized as the bastion of conventional crude oil and gas production, the area's exploration for natural resources –– in particular unconventional resources –– is in its infancy. The lack of fresh water may derail some of the exploration and production of unconventional resources in the Middle East. One of the solutions is to use the abundant availability of nearby sea water for fracturing treatments. This paper will discuss the applicability of sea water for fracturing fluids for without the need for separate treatment of the water. Rheological data with synthetic sea water as well as source sea water from Saudi Arabia, identification of any potential precipitation and remediation and compatibility with produced water and proppant pack conductivity data, of applicable fluids to show the effectiveness of the systems to the high temperatures of the reservoirs in the kingdom, 325°F will also be presented. The concept of using seawater as a base fluid is not new. Because of the problems associated with substituting seawater for freshwater in polymer-based fracturing fluids, many operators are apprehensive about using seawater for fracturing. There have been noted attempts to mix polymer-based fluids on the fly with seawater, but treatment results have varied widely. Seawater contains dissolved inorganic salts, adversely affecting hydration and viscosity development of polymer-based fluids. High content of calcium and magnesium in seawater can reduce viscosity. These salts also buffer and strongly influence pH control and may inhibit or deactivate certain gel breakers. To gel effectively, polymer fluids need a specific mixing environment with distinct pH windows. Borate crosslinking normally requires a high pH. Rheology and breaker profiles will be shown that provide the desired properties and regain conductivity to establish the non-damaging clean-up of a properly designed fluid. The technology presented uses chemical chelation of the problem ions in the sea water, resulting in the fracturing fluids with enhanced fluid and proppant pack properties, including thermal stability, retained fracture conductivity, pH buffering capacity, scale inhibition and fluid loss control. Further, the addition of the novel additives to the fluid does not interfere with the crosslink delay time and does not complicate the preparation of the fluid. The technology discussed eliminates the need for traditional water treatment and nano-filtration of sea water and associated disposal issues.

Fracturing fluids are most commonly aqueous systems comprising various polymers and crosslinking agents added to facilitate effective proppant transport and placement. A goal of fracturing practitioners has been to apply breaker technologies effective to eliminate or minimize the residual gel damage left behind by such systems in order to optimize the well stimulation. As a result of R&D efforts to that end, polymer-linkage-specific (polymer-specific) enzyme breakers were first introduced for fracturing applications in 1992. To date, these enzyme breakers have been employed in thousands of fracturing treatments around the world. Recent case studies evaluating long-term cumulative production have shown that wells in which polymer specific enzyme breakers have been applied demonstrate extraordinary performance when compared to production from offsets using other technologies. The long-term success of polymer-specific enzymes in fracturing operations for improving long-term production has been predicated upon a number of characteristics unique to enzymes, including their specificity to target polymer linkages and their functionality as catalysts which are not "spent" in the reactions they initiate. Additionally, contrary to popular misinformation, enzymes are effective at extremes of pH and temperature when properly applied. These unique properties are thought to make polymer-specific enzymes the polymer-degradation additive, which would produce the most effective proppant pack, thereby maximizing long-term productivity. The numerous case histories and offset comparisons documented in previous studies were, as a rule, relatively short-term in the prospective life of a well. The current endeavor seeks to provide detailed follow-up analyses of truly long-term production data from 170 fracture stimulated wells in the Canyon Sand, Penn, Lobo-Wilcox, Redfork, Hosston, and Grayburg/SanAndres formations and, the deep McKittrick field. Production histories over periods of up to 8 years were evaluated on wells treated with the polymer-specific enzymes and compared to offsets completed using conventional breaker technologies. The results clearly demonstrate the benefits of application of polymer-specific enzyme breakers on long-term well productivity. Introduction The enzymes historically used as breakers are non-specific mixtures that randomly hydrolyze polymers. These "conventional" enzymes are predominately mixtures of hemicellulase, cellulase, amylase and pectinase in unspecified ratios. Such enzymes are specific to react with guar, cellulose, starch and pectin polymers, respectively. All of these enzymes are hydrolases and, as such, are capable of binding with any of the aforementioned polymers. Since each of these enzymes is reactive with the linkages found in only one specific type of polymer, only the enzyme specific to that particular polymer will promote a cleavage. The other enzymes, once bound, can neither react with nor release from the polymer, effectively blocking the "right enzyme" from cleaving the polymer. This phenomena, known as competitive inhibition, results in the creation of polymeric fragments — generally the molecular weight of the polymer strand to which it is attached, plus the enzyme itself. In the case of crosslinked fluids, the "combined molecular weight" could be many times higher than the original molecular weight of the linear polymer due to crosslinking of the residual fragments. The result of this is a partial degradation of the polymer into predominately short- to medium-chain length polysaccharides, which are relatively insoluble and therefore may cause significant permeability damage.17–19 Polymer-specific enzymes were first introduced by Tjon-Joe-Pin and Brannon in 1992 for low-temperature, high-pH fracturing applications (60 - 140°F, pH 3–11) to affect improved cleanup of borate crosslinked fluids1. Since then more than 10,000 fracture stimulations have been performed on reservoirs with bottom-hole static temperatures (BHSTs) from 60°F to more than 300°F using guar-linkage-specific enzymes (GLSE). The GLSE complex consists of two isolated enzymes specific towards the linkages available between the sugar units of the guar polymer. Numerous research and laboratory studies have indicated this approach for degrading the guar polymer will inherently out-perform the conventionally used oxidative breakers (typically persulfate salts) and conventional non-specific enzyme mixtures.1–19

Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABA B receptor activation, receptors are recruited to the arrestin-channel complex and internalized. ␤-Arrestin 1 selectively binds to the SNAREbinding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.Inhibition of voltage-dependent calcium channels by seventransmembrane receptors (7TMR) 2 is one of the primary means of regulation of calcium-dependent physiological processes such as synaptic transmission, muscle contraction, and membrane excitability. In neurons, the Ca v 2.2 (N-type) channel is a prominent target for G protein-mediated modulation (1, 2). Inhibition of Ca v 2.2 channels can be voltage-dependent, and mediated by direct interactions with G protein ␤-␥ subunits (3, 4). In addition, kinases such as protein kinase C and tyrosine kinases have been shown to inhibit Ca v 2.2 channels in a voltage-independent manner (5, 6). Additional mechanisms may exist by which Ca 2ϩ influx is regulated. Dunlap and Fischbach (7) have suggested that transmitter-mediated shortening of the duration of the action potential could be due to a decrease in the number of voltage-dependent calcium channels at the membrane. Recently we have reported an additional mechanism by which 7TMRs can regulate neuronal calcium levels that involves a rapid internalization of voltage-dependent calcium channels into clathrin-coated vesicles upon receptor activation (8). Here we demonstrate that ␤-arrestin 1 is associated with Ca v 2.2 channels and that activation of 7TMRs results in the formation of an arrestin-receptor-channel complex. This interaction is required for internalization of calcium channels and plays a role in the modulation of calcium current. EXPERIMENTAL PROCEDURESMaterials-The following primary antibodies were used in these studies: rabbit anti-pan-␣ 1 (1:200,1.5 g/ml) (Alomone Labs, Jerusalem, Israel), anti-arrestin (1:500, BD Biosciences), and anti-GABAR1 (1:200, Chemicon). Anti-␤-arrestin 1 and anti-␤-arrestin 2 antibodies, and recombinant ␤-arrestin 1 and 2 (29) were kindly provided by the Lefkowitz laboratory. The following secondary antibodies were used in our studies: Oregon Green 488-conjugated goat anti-rabbit IgG (HϩL) (1:200, 10 g/ml), Cy3-conjugated goat anti-mouse IgG (HϩL) (1:200, 7.5 g/ml), and Cy5-goat anti-guinea pig IgG (HϩL) (1:200, 7.5 ...

Proppant diagenesis, as a topic of discussion in the industry, tends to generate negative reactions. Potential solutions, such as un-reactive coatings, cost money. Early research was done at substantially higher temperatures than many considered realistic, but was an attempt to shorten test times in order to begin to understand the phenomenon. Regardless of objections and negative reactions, the subject needs to be understood, because long term well performance implications may be significant.The purpose of this paper is to present a progress report on the results of long-term proppant diagenesis tests using coated and uncoated proppants, with and without shale reservoir rock present in the test cell, and in the presence of fluids of varying composition. Test philosophy and methods are described. Test results are documented using electron micrographs as well as chemical analysis and particle strength tests. Specific changes in fluid chemistry that result from contact with proppant and shale at reservoir temperature will also be a focus of the paper.