Palytoxin-induced Effects on Partial Reactions of the Na,K-ATPase

The interaction of palytoxin with the Na,K-ATPase was studied by the electrochromic styryl dye RH421, which monitors the amount of ions in the membrane domain of the pump. The toxin affected the pump function in the state P-E2, independently of the type of phosphorylation (ATP or inorganic phosphate). The palytoxin-induced modification of the protein consisted of two steps: toxin binding and a subsequent conformational change into a transmembrane ion channel. At 20°C, the rate-limiting reaction had a forward rate constant of 105 M−1s−1 and a backward rate constant of about 10−3 s−1. In the palytoxin-modified state, the binding affinity for Na+ and H+ was increased and reached values between those obtained in the E1 and P-E2 conformation under physiological conditions. Even under saturating palytoxin concentrations, the ATPase activity was not completely inhibited. In the Na/K mode, ∼50% of the enzyme remained active in the average, and in the Na-only mode 25%. The experimental findings indicate that an additional exit from the inhibited state exists. An obvious reaction pathway is a slow dephosphorylation of the palytoxin-inhibited state with a time constant of ∼100 s. Analysis of the effect of blockers of the extracellular and cytoplasmic access channels, TPA+ and Br2-Titu3+, respectively, showed that both access channels are part of the ion pathway in the palytoxin-modified protein. All experiments can be explained by an extension of the Post-Albers cycle, in which three additional states were added that branch off in the P-E2 state and lead to states in which the open-channel conformation is introduced and returns into the pump cycle in the occluded E2 state. The previously suggested molecular model for the channel state of the Na,K-ATPase as a conformation in which both gates between binding sites and aqueous phases are simultaneously in their open state is supported by this study

AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER). CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP) II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling

CNIH-2 is rendered a surface membrane protein by assembly with AMPARs.

<p><b>A</b> Total (T), surface (S) and internal (I) populations of CNIH-2 in HeLa cells expressing either CNIH-2 alone (CTRL) or together with GluA1_o or GluA2_i, respectively. S is concentrated 10fold. Note that in the absence of GluAs, CNIH-2 cannot be detected in the surface fraction. However, it is robustly observed in the plasma membrane when co-expressed with GluAs (n = 4). <b>B</b> Total (T), surface (S) and internal (I) populations of CNIH-2 in dissociated hippocampal neurons (DIV 17) transduced with CNIH-2 (+) or GFP (−). S is concentrated 10fold. Both endogenous (−) and over-expressed (+) CNIH-2 can be detected on the cell surface (n = 5). <b>C</b> (Top) Representative current traces recorded in somatic outside-out patches excised from dissociated hippocampal neurons (DIV 16–21) over-expressing either GFP (CTRL, black) or CNIH-2 (CNIH-2, red) upon 1 ms (left panel) and 100 ms applications (right panel) of 10 mM glutamate. (Bottom) Quantification of deactivation and desensitization kinetics. Data are given as mean ± SD. Asterisk denotes a significant difference from control (p<0.01, unpaired Student's t-test; deactivation: n = 10 and 8 for CTRL and CNIH-2, respectively; desensitization: n = 19 and 8 for CTRL and CNIH-2, respectively).</p

CNIH-2 facilitates ER export of AMPARs.

<p><b>A</b> Representative confocal images of OK cells stably expressing CNIH-2. Co-expression of dominant-negative Sar1 H79G prevents ER export of CNIH-2 leading to its redistribution into the ER. <b>B</b> Quantification of GluA1_o surface expression levels by extracellular epitope tagging in the presence of CNIH-2 and either wildtype (WT) Sar1 (white bar) or mutant Sar1 H79G (grey bar). Data are mean increases in surface expression levels by CNIH-2 ± SEM normalized to GluA1_o+Sar1 WT or GluA1_o+Sar1 H79G without CNIH-2, respectively. Asterisk marks a significant increase in surface expression of GluA1_o by co-expression of CNIH-2 (p<0.001, unpaired Student's t-test; n = 12 for both experimental groups). <b>C</b> Quantification of GluA1_o surface expression levels in the presence of CNIH-2 and either wildtype dynamin-1 (white bar) or dominant-negative dynamin-1 K44A (grey bar) inhibiting clathrin-dependent endocytosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030681#pone.0030681-Damke1" target="_blank">[38]</a>. Data are mean increases in surface expression levels by CNIH-2 ± SEM normalized as in B (n = 6 for both experimental groups).</p

CNIH-2 changes glycosylation of GluAs.

<p><b>A</b> Western blot analysis of total and surface populations of GluA2_i extracted from HeLa cells by surface biotinylation in the absence (CTRL) or presence of CNIH-2 (CNIH-2) (n = 4). Extensive glycosylation of surface GluA2_i during maturation increases its apparent molecular weight. Note the smaller increase upon co-expression of CNIH-2. <b>B</b> Enzymatic deglycosylation analysis of GluA2_i surface populations in the presence (+) or absence (−) of CNIH-2. Surface GluA2_i remained either untreated (C) or was incubated with either endoglycosidase H (H) or PNGase F (F). Note that upon CNIH-2 co-expression, the GluA2_i surface population remains sensitive to endoglycosidase H (n = 2).</p

Surface trafficking of GluAs by CNIH-2 is splicing-dependent.

<p><b>A</b> Quantification of GluA surface expression levels by extracellular epitope tagging in HeLa cells expressing the indicated GluA subunits. Data are mean ± SEM normalized to GluA1_o (GluA1_o: n = 24; GluA1_i: n = 9; GluA2_o: n = 12; GluA2_i: n = 12). <b>B</b> Increase in GluA surface expression mediated by CNIH-2 in HeLa cells. Data are mean ± SEM normalized to surface expression of respective GluA subunits without CNIH-2 (GluA1_o: n = 24; GluA1_i: n = 8; GluA2_o: n = 12; GluA2_i: n = 12).</p