Intact Cl Research Articles

Calcineurin Inhibition Impairs the Function of Neuronal Potassium‐Chloride Cotransporter 2

Calcineurin inhibitors (CnI) such as Cyclosporin A (CsA) are instrumental for immunosuppression after organ transplantation but may cause serious neurologic side effects, including seizures.Neuronal excitability depends on intact Cl− homeostasis. Generation of hyperpolarizing synaptic responses to GABA and glycine requires a Cl− gradient across the cell membrane with a low [Cl−]i, mainly established by K+‐Cl− cotransporter 2 (KCC2) and to a lesser extent Na+‐K+‐Clcotransporter 1 (NKCC1); both are cation‐coupled chloride cotransporters (CCCs). Calcineurin has been implicated in the regulation of CCCs, whereas impaired CCC function is a well‐known condition in human pharmacoresistant epilepsy. Therefore, we hypothesized that CsA affects KCC2 or NKCC1 functions, thus causing neuronal hyperexcitability.In ex vivo intracellular recordings with sharp microelectrodes, Wistar rat pyramidal neurons in neocortical slices responded to CsA (5 μM for 1h) with a less negative GABAA reversal potential (+7.2 mV) and prolonged Cl− extrusion time after iontophoretic Cl− loading (+3.9 s). 2‐photon fluorescence lifetime imaging in presence of Cl− sensitive dye MQAE showed an increased [Cl−]i in layer V neurons (+6.9 mM), suggesting reduced KCC2 activity. Co‐immunoprecipitation studies in rodent brain tissue suggested interactions of calcineurin Aβ (CnAβ) with KCC2 and NKCC1.In vivo, CsA administration to rats (5–25 mg/kg i.p.) enhanced levels of inhibitory tyrosine KCC2 phosphorylation at short term (4h; +172%) and reduced levels of activating S940‐KCC2 phosphorylation in the long term (14d; −61%). Reduced phospho‐S940‐KCC2 levels were observed in further models of calcineurin inhibition such as genetic CnAβ‐deficiency in mice (−78%) or CsA treatment of zebrafish larvae (10 μM in water: −54% after 24h). In contrast, NKCC1 and its activating kinase SPAK were stimulated upon calcineurin inhibition in rodents. Similar data were obtained in Drosophila melanogaster.Our results provide evidence that CnI attenuate KCC2 function but may stimulate NKCC1 leading to elevated [Cl−]i. These effects may increase neuronal excitability and contribute to CnI neurological adverse effects. The benefits of KCC2 activators or SPAK inhibitors for enhancing chloride extrusion in patients experiencing CnI neurotoxicity deserve further characterization.Support or Funding InformationThis study was supported by Deutsche Forschergemeinschaft.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simple, Direct Routes to Polymer Brush Traps and Nanostructures for Studies of Diffusional Transport in Supported Lipid Bilayers.

Patterned poly(oligo ethylene glycol) methyl ether methacrylate (POEGMEMA) brush structures may be formed by using a combination of atom-transfer radical polymerization (ATRP) and UV photopatterning. UV photolysis is used to selectively dechlorinate films of 4-(chloromethyl)phenyltrichlorosilane (CMPTS) adsorbed on silica surfaces, by exposure either through a mask or using a two-beam interferometer. Exposure through a mask yields patterns of carboxylic acid-terminated adsorbates. POEGMEMA may be grown from intact Cl initiators that were masked during exposure. Corrals, traps, and other structures formed in this way enable the patterning of proteins, vesicles, and, following vesicle rupture, supported lipid bilayers (SLBs). Bilayers adsorbed on the carboxylic acid-terminated surfaces formed by C–Cl bond photolysis in CMPTS exhibit high mobility. SLBs do not form on POEGMEMA. Using traps consisting of carboxylic acid-functionalized regions enclosed by POEGMEMA structures, electrophoresis may be observed in lipid bilayers containing a small amount of a fluorescent dye. Segregation of dye at one end of the traps was measured by fluorescence microscopy. The increase in the fluorescence intensity was found to be proportional to the trap length, while the time taken to reach the maximum value was inversely proportional to the trap length, indicating uniform, rapid diffusion in all of the traps. Nanostructured materials were formed using interferometric lithography. Channels were defined by exposure of CMPTS films to maxima in the interferogram, and POEGMEMA walls were formed by ATRP. As for the micrometer-scale patterns, bilayers did not form on the POEGMEMA structures, and high lipid mobilities were measured in the polymer-free regions of the channels.

Facilitators of a Proton-Glutamate in a Cl -/H + Exchanger, CLC-EC1

The CLC-ec1 is a bacterial homologue of the CLC Cl-/H+ exchanger that catalyzes 2:1 exchange of Cl- and H+. Functionally intact monomers form a dimer in bilayer membranes. Gating-glutamate (or Gluex, E148) is the pH-dependent external gate of both Cl- and H+ transport and proton-glutamate (or Gluin, E203) mediates proton-transport from intracellular solution to the protein interior. Mutations on either glutamate with non-protonable amino acids abolish H+ transport across the membrane. However, it is still puzzling how protons in the intracellular solution could be transferred to the proton-gluatamate forming a salt bridge with arginine (R28) in swapped N-terminal domain from neighboring subunit, which structurally isolates the proton-glutamate from the intracellular solution. In order to find functional facilitators for proton transport, we have been examining several residues near E203. Salt-bridge breaking mutants, R28L, Q and E do not alter the functional activity at all. Moreover, N-terminal truncation mutants (Delta16 and Delta29), which remove an apparent “lid” covering the proton-glutamate, preserve intact Cl-/H+ exchanger activity. Mutations on two proximate glutamates (E113L and E202L) decrease H+ transport rate without changing Cl- turnover rate. Currently, we are investigating functional and structural changes of multiple mutations.

Nutritional and Hormonal Factors Influencing Follicle Development in Cattle

Ovarian antral follicular development in cattle proceeds through stages of follicular recruitment, selection and dominance. This is a reoccurring process during the estrous cycle when either one or two dominant anovulatory follicles develop prior to development of the ovulatory follicle. These changes in follicle development can be monitored by ultrasonography (Savio et al, 1988: Sirois and Fortune, 1988; Knopf et al, 1990). Apparently, dominant follicles control development of other follicles through the production of factors that act locally and/or systemically. This regulatory process is currently under intensive investigation. The process of dominance causes a single ovulation which is characteristic of cattle. We believe that growth of anovulatory dominant follicles during the estrous cycle is halted due to the negative feedback effect of progesterone from the CL on LH secretion. Insufficient LH secretion will not support the continued growth and function of the follicle. Consequently, the dominant follicle undergoes atresia and a new follicle is recruited and becomes the dominant follicle of the next wave. The importance of LH secretion was tested (Savio et al, 1990a) by giving PGF2 " (Lutalyse; Upjohn Co., Kalamazoo, MI) on day 8 of the estrous cycle and maintaining a weak progestin environment by the insertion of a norgestomet implant into the ear (Synchromate-B; Ceva Laboratories, Overland Park, KS). The implant was either removed on day 23 post estrus (Treatment 1) or replaced with a new implant on day 18 which also was removed on day 23 (Treatment 2). It is clear that the first wave follicle was sustained until the implant was removed on day 23 (Figure 1 ). Furthermore, growth of this single follicle wave was greater in cows of treatment 1 that retained the original implant. Indeed basal LH secretion was greater on days 10 (5 pulses per 8 hours) and 19 (7 pulses per 8 hours) postestrus in treatment 1 compared to day 10 of the estrous cycle ( control animals with an intact CL; .75 pulses per 8 hours). Insertion of a new implant on day 18 (treatment 2) reduced the pulse frequency of LH on day 19 (3 pulses per 8 hours). This experimental model demonstrated that alterations in basal LH secretion modulate follicular development during the estrous cycle. This has profound implications relative to the management of ovarian follicles for the synchronization of estrus and subsequent fertility at this estrus. Furthermore, this endocrine scenario may account for the occurrence of ovarian follicular cysts in the early postpartum period. Indeed the early postpartum period is characterized by early growth of ovarian follicles in the absence of a CL (low progesterone; Savio et al., 1990b ). Perhaps a sustained basal secretion of LH and the inability to have a preovulatory surge of LH (hypothalamic-pituitary dysfunction) leads to the development of dominant and estrogenic follicular cysts. Our basic understanding of ovarian follicular dynamics will lead to rationale systems of follicular management to improve reproductive efficiency.

CI Inhibitor: Different Mechanisms of Reaction with Complement Component Ci¯t and Ci¯ts

Inactivation of human complement subcomponent C1-s by its regulator C1 inhibitor at physiological ionic strength proceeded at a 3-fold higher rate when C1-s was in the physiological C1- complex with subcomponents C1q and C1-r rather than as purified subunit. When the C1- complex was disassembled by chelation of calcium, the C1-s subcomponent was inactivated by C1 inhibitor at rates similar to those for the purified proteinase. Increasing ionic strength had little effect on the reaction of purified C1-s with C1 inhibitor but greatly diminished the rate of reaction of intact C1-. Addition of heparin accelerated the inactivation of purified C1-s by C1 inhibitor up to 25-fold but increased the inactivation of intact C1- only about 5-fold. These differences in the inactivation of C1-s by C1 inhibitor, depending on whether the proteinase is free or complexed with other subcomponents of C1-, suggest different mechanisms of reaction. Occurrence of subcomponent C1-s in a macromolecular complex with C1q and C1-r, thus, appears to be critical not only for directing its physiological activation but also its inactivation.

Fluorescence of the tryptophyl residues of the constant fragment of the immunoglobulin light chain.

The constant (CL) domain of the type-lambda immunoglobulin light chain contains two tryptophyl residues. The fluorescence lifetimes of the CL fragment and its two modified forms, the reduced CL, and reduced and alkylated CL fragments, were measured at pH 7.5 and 25 degrees C. The fluorescence decay kinetics of these fragments were described in terms of two lifetimes. The static and dynamic quenching reactions by acrylamide of the tryptophyl residues of these fragments were measured and their dynamic properties were compared with the static properties reported previously (Goto, Y. & Hamaguchi, K. (1979) J. Biochem. 86, 1433-1441; Goto, Y. & Hamaguchi, K. (1986) Biochemistry 25, 2821-2828; Ashikari, Y., Arata, Y., & Hamaguchi, K. (1985) J. Biochem. 97, 517-528). It was found that although the static conformation of the reduced CL fragment is very similar to that of the intact CL fragment, the dynamic conformations of these two fragments differ considerably.

Conformation of the constant fragment of the immunoglobulin light chain: effect of cleavage of the polypeptide chain and the disulfide bond.

In order to understand the conformations and stabilities of the immunoglobulin domains, a derivative of the type-lambda constant fragment in which the peptide bond Arg190-Ser191 is cleaved and a derivative of the type-kappa constant fragment in which the peptide bond Arg142-Glu143 is cleaved were prepared by limited proteolysis with the proteinase from mouse submaxillary gland, endoproteinase Arg-C [EC 3.4.21.40]. The cleaved peptide bond of each derivative is located in the loop formed by the intrachain disulfide bond and the two peptides formed are linked by the disulfide bond. The two nicked CL fragments did not assume any ordered conformation. On the other hand, a derivative in which the intrachain disulfide bond is reduced had a conformation very similar to that of the intact CL fragment, although the stability was considerably low. Since the entropy of the nicked fragment should be nearly the same as that of the reduced CL fragment in the unfolded state, a destabilizing effect of cleavage of the polypeptide bond in the folded state in addition to the entropic effect in the unfolded state seems necessary to account for the conformations of the nicked CL fragments.

Conformation and stability of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge.

The constant fragment of the immunoglobulin light chain in which the intramolecular disulfide bond is reduced (reduced CL fragment) assumes a conformation very similar to that of the intact CL fragment and contains two sulfhydryl groups buried in the interior of the molecule [Goto, Y., & Hamaguchi, K. (1979) J. Biochem. (Tokyo) 86, 1433-1441]. In order to understand the role of the disulfide bond, a derivative in which the disulfide bond is replaced by an S-Hg-S bond was prepared and its conformation and stability were studied. The derivative was prepared by reacting the reduced CL fragment with mercuric chloride. Kinetic studies showed that the reaction is rate-limited by the unfolding process of the reduced CL fragment. The mercury derivative was as compact as the intact CL or reduced CL fragment, and a tryptophyl residue was found to be buried near the S-Hg-S bond in the interior of the protein molecule. Judging from the circular dichroic spectrum, however, the beta-structure characteristic of the immunoglobulin fold was disturbed. The stability of the derivative to guanidine hydrochloride was lower than that of the intact CL fragment, but the unfolding transition was reversible and cooperative. Decreased stability of the mercury derivative is due to its folded conformation being distorted by introduction of the S-Hg-S bond.

Unfolding and refolding of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge.

The conformation of the constant fragment of the immunoglobulin light chain in which the intrachain disulfide bond is replaced by the bond S-Hg-S (CL-Hg fragment), was as compact as that of the intact CL fragment, but its stability to guanidine hydrochloride was lower than that of the intact CL fragment [Goto, Y. & Hamaguchi, K. (1986) Biochemistry in press]. The kinetics of reversible unfolding and refolding of the CL-Hg fragment by guanidine hydrochloride were studied and compared with those for the intact CL and reduced CL fragments [Goto, Y. & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910, 911-926]. The unfolding kinetics were explained on the basis of a three-species mechanism, U1----U2----F, where U1 and U2 are respectively slow-folding and fast-folding species of unfolded protein, and F is folded protein. However, an additional isomerization, though its contribution to the overall reaction process is small, had to be taken into account to explain the refolding kinetics. The kinetic properties of interconversion between U1 and U2 were similar to those for the intact CL and reduced CL fragments. This suggested that the same prolyl residue is involved in the isomerization reactions in the unfolded states of the intact CL, reduced CL, and CL-Hg fragments. The rate constant for the unfolding process, F to U2, was about 20 times greater than those for the intact CL and reduced CL fragments, while the rate constant for the refolding process, U2 to F, lay between the values for the intact CL and the reduced CL fragment. The free energy profiles of unfolding and refolding of the intact CL, reduced CL, and CL-Hg fragments were compared.

PH-induced unfolding of the constant fragment of the immunoglobulin light chain: effect of reduction of the intrachain disulfide bond.

Circular dichroism and proton nuclear magnetic resonance measurements were made to clarify how reduction and reduction plus alkylation of the intrachain disulfide bond affect the conformation and stability of the constant (CL) fragment of the immunoglobulin light chain. The pH titration behavior of the two histidine residues in the intact CL, reduced CL, and reduced and alkylated CL fragments were followed by proton nuclear magnetic resonance spectroscopy. It was shown that reduction of the intrachain disulfide bond does not affect the solution conformation of the immunoglobulin fold, whereas reduction and alkylation results in extensive unfolding of the protein molecule. These results are consistent with the previous results obtained by Goto and Hamaguchi [1979) J. Biochem. 86, 1433-1441) using circular dichroism, fluorescence, and titration of SH groups. The pH-induced unfolding of the reduced CL fragment was compared with that of the intact CL fragment. The stability of the intact CL fragment to acid was much greater than that of the reduced CL fragment. The equilibria of the unfolding by acid could be explained by assuming that the same ionizable groups participate in the unfolding for the reduced CL fragment as for the intact CL fragment, and that the stability of the intact CL fragment is 100 times greater than that of the reduced CL fragment. In the alkaline pH region, the reduced CL fragment was unfolded above pH 8.5, while the intact CL fragment was not unfolded until pH 11.6. The kinetics of the unfolding by alkali of the reduced CL fragment was also studied. The unfolding by alkali of the reduced CL fragment could be explained by assuming that the reduced CL molecule can no longer adopt the folded conformation when either or both SH groups ionize.

The role of the intrachain disulfide bond in the conformation and stability of the constant fragment of the immunoglobulin light chain.

The conformation and stabilities of the CL fragment isolated from a type lambda Bence Jones protein and the fragment in which the intrachain disulfide bond had been reduced were studied by measuring CD, fluorescence, and ultraviolet absorption. The results indicated that no great conformational change occurs on reduction of the disulfide, unless the SH groups are alkylated. Intact CL was more resistant than reduced CL to guanidine hydrochloride. The denaturation curves were analyzed using an equation based on the binding of guanidine hydrochloride and the free energy changes of denaturation in the absence of the denaturant were estimated as about 6 kcal.mol-1 for intact CL and about 1.8 kcal.mol-1 for reduced CL. The difference in stability between intact CL and reduced CL was explained to a great extent in terms of the entropy change associated with reduction of the intrachain disulfide bond of the fragment in the denatured state.