Electrogenic Kinetics of a Mammalian Intestinal Type IIb Na+/Pi Cotransporter

The kinetics of a type IIb Na(+)-coupled inorganic phosphate (Pi) cotransporter (NaPi-IIb) cloned from mouse small intestine were studied using the two-electrode voltage clamp applied to Xenopus oocytes. In the steady state, mouse NaPi-IIb showed a curvilinear I-V relationship, with rate-limiting behavior only for depolarizing potentials. The Pi dose dependence was Michaelian, with an apparent affinity constant for Pi (Km(pi)) of 10 +/- 1 microM: at -60 mV. Unlike for rat NaPi-IIa, (Km(pi)) increased with membrane hyperpolarization, as reported for human NaPi-IIa, flounder NaPi-IIb and zebrafish NaPi-IIb2. The apparent affinity constant for Na(+) (Km(na)) was 23 +/- 1 mM: at -60 mV, and the Na(+) activation was cooperative with a Hill coefficient of approximately 2. Pre-steady-state currents were documented in the absence of Pi and showed a strong dependence on external Na(+). The hyperpolarizing shift of the charge distribution midpoint potential was 65 mV/log[Na]. Approximately half the moveable charge was attributable to the empty carrier. A comparison of the voltage dependence of steady-state Pi-induced current and pre-steady-state charge movement indicated that for -120 mV 相似文献   

The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix

The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.     

Systematic review and meta-analysis of the efficacy and safety of existing TNF blocking agents in treatment of rheumatoid arthritis

Background and Objectives

Five-tumour necrosis factor (TNF)-blockers (infliximab, etanercept, adalimumab, certolizumab pegol and golimumab) are available for treatment of rheumatoid arthritis. Only few clinical trials compare one TNF-blocker to another. Hence, a systematic review is required to indirectly compare the substances. The aim of our study is to estimate the efficacy and the safety of TNF-blockers in the treatment of rheumatoid arthritis (RA) and indirectly compare all five currently available blockers by combining the results from included randomized clinical trials (RCT).

Methods

A systematic literature review was conducted using databases including: MEDLINE, SCOPUS (including EMBASE), Cochrane library and electronic search alerts. Only articles reporting double-blind RCTs of TNF-blockers vs. placebo, with or without concomitant methotrexate (MTX), in treatment of RA were selected. Data collected were information of patients, interventions, controls, outcomes, study methods and eventual sources of bias.

Results

Forty-one articles reporting on 26 RCTs were included in the systematic review and meta-analysis. Five RCTs studied infliximab, seven etanercept, eight adalimumab, three golimumab and three certolizumab. TNF-blockers were more efficacious than placebo at all time points but were comparable to MTX. TNF-blocker and MTX combination was superior to either MTX or TNF-blocker alone. Increasing doses did not improve the efficacy. TNF-blockers were relatively safe compared to either MTX or placebo.

Conclusions

No single substance clearly rose above others in efficacy, but the results of the safety analyses suggest that etanercept might be the safest alternative. Interestingly, MTX performs nearly identically considering both efficacy and safety aspects with a margin of costs.     

Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe

We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3' and 5' directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage (Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3- -induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).     

Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation

While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.     

Functional characterization of human NBC4 as an electrogenic Na+-HCO3- cotransporter (NBCe2)

We havefunctionally characterized Na⁺-driven bicarbonatetransporter (NBC)4, originally cloned from human heart by Pushkin etal. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the fourNBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no.AF293337) in Xenopus laevis oocytes and assayed membrane potential (V_m) and pH regulatory function withmicroelectrodes. Exposing an NBC4c-expressing oocyte to a solutioncontaining 5% CO₂ and 33 mM HCOelicited a large hyperpolarization, indicating that the transporter iselectrogenic. The initial CO₂-induced decrease inintracellular pH (pH_i) was followed by a slow recovery thatwas reversed by removing external Na⁺. Two-electrodevoltage clamp of NBC4c-expressing oocytes revealed largeHCO- and Na⁺-dependent currents. When wevoltage clamped V_m far from NBC4c's estimatedreversal potential (E_rev), the pH_irecovery rate increased substantially. Both the currents andpH_i recovery were blocked by 200 µM4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimatedthe transporter's HCO:Na⁺ stoichiometryby measuring E_rev at different extracellularNa⁺ concentration ([Na⁺]_o)values. A plot of E_rev againstlog[Na⁺]_o was linear, with a slope of 54.8 mV/log[Na⁺]_o. This observation, as well asthe absolute E_rev values, are consistent with a2:1 stoichiometry. In conclusion, the behavior of NBC4c, which wepropose to call NBCe2-c, is similar to that of NBCe1, the firstelectrogenic NBC.

     

Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches

Shearzyme (GH10 endo-1,4-β-d-xylanase) and two different α-l-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with α-l-Araf (1→2)-monosubstituted β-d-Xylp residues or α-l-Araf (1→2)- and (1→3) doubly substituted β-d-Xylp residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp, d-II: α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp-(1→4)-d-Xylp, m-I: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-β-d-Xylp-(1→4)-d-Xylp and m-II: α-l-Araf-(1→2)-β-d-Xylp-(1→4)-d-Xylp. To our knowledge, this is the first report on structural ¹H and ¹³C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly α-l-Araf (1→2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.     

Manipulating the genetic code for membrane protein production: What have we learnt so far?

With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production. This article is part of a Special Issue entitled: Protein Folding in Membranes.