Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

Swick, Andrew G.; Janicot, Michel; Cheneval-Kastelic, T; McLenithan, John C.; Lane, M. Daniel

doi:10.1073/pnas.89.5.1812

Cited by 117 publications

(83 citation statements)

References 22 publications

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“…Using a Drummond Nanoject (Drummond Scientific, Broomall, PA), 4.7 nl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) containing 0.15 ng of PMT3-rSGLT1 or PMT3-A166C-rSGLT1 and 0.15 ng of PMT3-SEAP was injected into the animal pole of the defolliculated oocytes as described previously by Swick et al (17). The injected oocytes were kept in MBS supplemented with 2.5 mM sodium pyruvate for 2-3 days before transfer to 96-well plates and incubation individually another 16 -24 h. The incubation solution from each oocyte was then tested for alkaline phosphatase activity following the protocol of Tate et al (18).…”

Section: Methodsmentioning

confidence: 99%

Replacement of Ala-166 with Cysteine in the High Affinity Rabbit Sodium/Glucose Transporter Alters Transport Kinetics and Allows Methanethiosulfonate Ethylamine to Inhibit Transporter Function

Lo¹,

Silverman

1998

Journal of Biological Chemistry

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An alanine to cysteine mutation at position 166 has been introduced by site-directed mutagenesis into the rabbit sodium/glucose transporter (rSGLT1). When expressed in Xenopus laevis oocytes, this mutant transporter (A166C rSGLT1) demonstrates a significantly lower apparent affinity for ␣-methyl glucoside (␣MG) compared with the wild-type transporter (apparent K m ‫؍‬ 0.8 versus 0.15 mM). Using the two-electrode voltage clamp technique, transient currents have also been measured, and for the mutant transporter, the transients induced by large depolarizations exhibit longer time constants than those for wild type. Moreover, the substitution of Ala-166 with a cysteine allows the sulfydryl specific reagent, methanethiosulfonate ethylamine (MTSEA), to react with and alter the function of the transporter. Whereas the wild-type transporter is unaffected by reaction with MTSEA, A166C rSGLT1 has its steady-state currents induced by 1 mM ␣MG inhibited 83% within a minute of exposure to MTSEA. Furthermore, the pre-steady-state transients of the A166C mutant after MTSEA exposure demonstrate much shorter time constants than before while the total amount of charge transferred is only slightly diminished. These results together provide evidence that position 166 is situated in a region critical to the functioning of rSGLT1.

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Section: Methodsmentioning

confidence: 99%

Replacement of Ala-166 with Cysteine in the High Affinity Rabbit Sodium/Glucose Transporter Alters Transport Kinetics and Allows Methanethiosulfonate Ethylamine to Inhibit Transporter Function

Lo¹,

Silverman

1998

Journal of Biological Chemistry

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“…Oocytes-Human ASIC1a-and ASIC2a-encoding cDNAs in the pMT3 mammalian expression vector have been described previously (19,20). These sequences correspond to GenBank TM accession number NM_001095 for human ASIC1a and NM_001094 for human ASIC2a.…”

Section: Construction Of Chimeras Site-directed Mutagenesis and Expmentioning

confidence: 99%

Identification of Protein Domains That Control Proton and Calcium Sensitivity of ASIC1a

Sherwood

Franke

Conneely

et al. 2009

Journal of Biological Chemistry

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The acid-sensing ion channels (ASICs) open in response to extracellular acidic pH, and individual subunits display differential sensitivity to protons and calcium. ASIC1a acts as a high affinity proton sensor, whereas ASIC2a requires substantially greater proton concentrations to activate. Using chimeras composed of ASIC1a and ASIC2a, we determined that two regions of the extracellular domain (residues 87-197 and 323-431) specify the high affinity proton response of ASIC1a. These two regions appear to undergo intersubunit interactions within the multimeric channel to specify proton sensitivity. Single amino acid mutations revealed that amino acids around Asp 357 play a prominent role in determining the pH dose response of ASIC1a. Within the same region, mutation F352L abolished PcTx1 modulation of ASIC1a. Surprisingly, we determined that another area of the extracellular domain was required for calcium-dependent regulation of ASIC1a activation, and this region functioned independently of high affinity proton sensing. These results indicate that specific regions play overlapping roles in pH-dependent gating and PcTx1-dependent modulation of ASIC1a activity, whereas a distinct region determines the calcium dependence of ASIC1a activation.The acid-sensing ion channels (ASICs) 3 are proton-gated ion channels expressed in neurons throughout the central and peripheral nervous system (1-3). ASICs are activated by extracellular acidosis, and protons act as ligands triggering channel opening (4). Disruption of the accn2 gene (which encodes ASIC1) dramatically reduces proton-gated currents in central neurons and alters a variety of behaviors, including fear, learning, and memory (5, 6). ASIC1 also contributes to neuronal damage and death during the prolonged acidosis accompanying cerebral ischemia (7). Specifically, mice with disruptions in the accn2 gene display 60% smaller lesion size compared with normal mice in models of stroke (8). Application of PcTx1, a venom peptide that prevents ASIC1a activation, is similarly neuroprotective, even when administered hours after injury (8, 9). Thus, ASIC1a represents a novel pharmacological target for the prevention of neuronal death following stroke.Mammals have four genes encoding ASICs (accn1 to -4) that encode at least six different ASIC subunits (1-3, 10). Like all members of the DEG/ENaC family, individual ASIC subunits have two transmembrane regions separated by a large cysteinerich extracellular region. Three ASIC subunits associate to form homomeric or heteromeric channels with distinct biophysical characteristics (11)(12)(13)(14). Specifically, ASIC1a homomeric channels activate at pH values much closer to neutral pH compared with ASIC2a homomeric channels. The high affinity proton sensitivity of ASIC1a plays a prominent role in acidosisinduced neuronal death, and modulators that alter the pH dose response of ASIC1a affect neuronal sensitivity to prolonged acidosis (8, 9, 15). For example, the neuroprotective venom peptide PcTx1 increases the proton sensitivity of the ...

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“…Aliquots of each plasmid preparation were diluted to 65 ng/pl in 50 mM KCI, 50 mM KH2P04, pH 7.6, containing 6.5 ng/,U of a recombinant pMT21 bearing cDNA for expression of secreted alkaline phosphatase (pMT-SEAP) [13] for injection into oocytes.…”

Section: Oocyte Injection and Maintenancementioning

confidence: 99%

Electrogenic amino acid exchange via the rBAT transporter

et al. 1994

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A cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

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Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

Cited by 117 publications

References 22 publications

Replacement of Ala-166 with Cysteine in the High Affinity Rabbit Sodium/Glucose Transporter Alters Transport Kinetics and Allows Methanethiosulfonate Ethylamine to Inhibit Transporter Function

Replacement of Ala-166 with Cysteine in the High Affinity Rabbit Sodium/Glucose Transporter Alters Transport Kinetics and Allows Methanethiosulfonate Ethylamine to Inhibit Transporter Function

Identification of Protein Domains That Control Proton and Calcium Sensitivity of ASIC1a

Electrogenic amino acid exchange via the rBAT transporter

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