PVP‐containing solutions for analysis of divalent cation‐dependent NMDA responses in <i>Xenopus</i> oocytes

Raditsch, Martin; Witzemann, Veit

doi:10.1016/0014-5793(94)01118-4

Cited by 13 publications

(12 citation statements)

References 18 publications

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“…As previously reported by Raditsch and Witzemann (1994), addition of 2 mM PVP to EGTA-containing, divalent-free Ringer's solution greatly reduced the membrane leak current and allowed for stable measurements of NMDA-induced currents to be made under these conditions. Figure 1 shows the effects of 100 mM ethanol on NR1/2A receptor currents measured in divalent cation-free PVP-Ringer' s solution.…”

Section: Ethanol Sensitivity Of Nmda Receptors In the Absence Of Divamentioning

confidence: 85%

Intracellular Calcium Enhances the Ethanol Sensitivity of NMDA Receptors Through an Interaction with the C0 Domain of the NR1 Subunit

Mirshahi

Anders

Ronald

et al. 1998

Journal of Neurochemistry

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Previous studies in this laboratory have shown that the ethanol inhibition of recombinant NMDA receptors expressed in Xenopus oocytes is subunit‐dependent, with the NR1/2A receptor being more sensitive than NR1/2C receptors. The ethanol sensitivity of NR1/2A receptors is reduced by substitution of the wild‐type NR1‐1a (NR1011) subunit with the calcium‐impermeable NR1 (N616R) subunit. In the present study, the ethanol inhibition of NMDA receptors was determined under different conditions to examine the role that calcium plays in determining the ethanol sensitivity of recombinant NMDA receptors. The ethanol sensitivity of NR1/2B or NR1/2C receptors was not affected by alterations in extracellular calcium levels or by coexpression with calcium‐impermeable NR1 mutants. In contrast, the inhibition of NR1/2A receptors by 100 mM ethanol was reduced in divalent‐free recording medium and was significantly increased when 10 mM calcium was used as the only charge carrier. The increase in the ethanol sensitivity of NR1/2A receptors under high‐calcium conditions was prevented by preinjection of oocytes with the calcium chelator 1,2‐bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) but not by inhibitors of calmodulin or protein kinase C. Ethanol did not alter the channel blocking activity of divalent cations on NMDA‐induced currents. The enhanced ethanol sensitivity of NR1/2A receptors in 10 mM calcium persisted when the NR1 subunit was replaced by the alternative splice variant NR1‐4a (NR1000), which lacks the C1 and C2 cassettes. However, expression of a mutant NR1 subunit that lacked the C0, C1, and C2 domains abolished the calcium‐dependent enhancement of ethanol's inhibition of NR1/2A receptors. Finally, the ethanol sensitivity of wild‐type NR1/2A receptors measured in transfected HEK 293 cells by whole cell patch‐clamp electrophysiology was significantly reduced by expression of the C‐terminal truncated NR1 subunit. These results demonstrate that the ethanol sensitivity of certain NMDA receptors is modulated by an intracellular, calcium‐dependent process that requires the C0 domain of the NR1 subunit.

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Section: Ethanol Sensitivity Of Nmda Receptors In the Absence Of Divamentioning

confidence: 85%

Intracellular Calcium Enhances the Ethanol Sensitivity of NMDA Receptors Through an Interaction with the C0 Domain of the NR1 Subunit

Mirshahi

Anders

Ronald

et al. 1998

Journal of Neurochemistry

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“…3 C ) . Removal of the respective antagonist from the bathing solution revealed that receptors recovered (Raditsch and Witzemann, 1994). Stopping application of antagonist Me-D-AspR-mediated currents blocked by argiotoxin,,, (100 nM) recovered within 3 min up to 72% (a) whereas channels blocked by MK-801 (100 nM) showed only 27 % recovery during the same tiine (c).…”

Section: Resultsmentioning

confidence: 99%

“…Ba2+ (Leonard and Kelso, 1990) and polyvinylpyrrolidone 25, (Raditsch and Witzemann, 1994), were used in order to reduce Ca2+-evoked chloride currents in oocytes. Both agonists, glutamate (0.1 mM) and glycine (0.01 mM), were dissolved either in normal frog Ringer (NFR), or in modified, nominal Ca2+-free buffers such as BaZ+FR described above.…”

Section: Methodsmentioning

confidence: 99%

Polyamine Spider Toxins and Mammalian N‐Methyl‐d‐Aspartate Receptors

Raditsch

Geyer

Kalbitzer

et al. 1996

European Journal of Biochemistry

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Recombinant N-methyl-D-aspartate receptors composed of NRlNR2A subunits were expressed in Xenopus oocytes to analyse the voltage-dependent and use-dependent channel blocking activity of argiotoxin,,,. Functional assays demonstrate that the toxin competes with other open channel blockers such as MgZ+ and MK-801. Direct binding or competition assays using radiolabeled ligands and isolated rat brain membranes, in contrast, reveal no specific binding or yield binding constants which differ by orders of magnitude from the IC,, values of the functional assays. One explanation is that argiotoxin,,, does not bind with high affinity to the inhibitory site in the N-methyl-D-aspartate-receptor channel under in vitro conditions when membranes are depolarised. The structure of argiotoxin63, was investigated by NMR spectroscopy. In solution the positively charged argiotoxin,,, acquires an extended conformation and its dimensions might allow permeation deep into the channel. In the absence of direct structural information on the channel protein, the detailed analysis of blockade in conjunction with structural information, as provided here, may be of aid in the deduction of structural features of glutamate-receptor channel ion pores.

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“…They are expressed in a broad variety of tissues and known to mediate fast synaptic transmission [7,8] and synaptic transmission between sympathetic nerves and smooth muscle [9,19]. By now 7 P2X receptor subunits (P2X!-P2X 7 ) have been cloned which share a putative two-transmembrane segment structure with intracellular C-and N-termini and an extracellular loop [2,3,5,6,14,20,21]. Heterologous expression of each subunit results in the formation of non-selective cation channels with differential sensitivities for the agonists ATP and a,|3-methylene-ATP and the antagonists pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin.…”

Section: Introductionmentioning

confidence: 99%

Desensitization of the P2X₂ receptor controlled by alternative splicing

et al. 1997

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P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X 2 receptor splice variant (P2X2_2) carrying a 207 bp deletion in the intracellular Cterminus and the analysis of the corresponding genomic structure of the P2X 2 gene. P2X 2 _2 is as highly expressed as the original P2X 2 sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X 2 or P2X2_ 2 receptors showed significant differences in desensitization time constants and steady-state currents in the continuous presence of ATP. These results imply functional differences between cells differentially expressing these P2X 2 isoforms.

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PVP‐containing solutions for analysis of divalent cation‐dependent NMDA responses in Xenopus oocytes

Cited by 13 publications

References 18 publications

Intracellular Calcium Enhances the Ethanol Sensitivity of NMDA Receptors Through an Interaction with the C0 Domain of the NR1 Subunit

Intracellular Calcium Enhances the Ethanol Sensitivity of NMDA Receptors Through an Interaction with the C0 Domain of the NR1 Subunit

Polyamine Spider Toxins and Mammalian N‐Methyl‐d‐Aspartate Receptors

Desensitization of the P2X₂ receptor controlled by alternative splicing

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PVP‐containing solutions for analysis of divalent cation‐dependent NMDA responses in Xenopus oocytes

Cited by 13 publications

References 18 publications

Intracellular Calcium Enhances the Ethanol Sensitivity of NMDA Receptors Through an Interaction with the C0 Domain of the NR1 Subunit

Intracellular Calcium Enhances the Ethanol Sensitivity of NMDA Receptors Through an Interaction with the C0 Domain of the NR1 Subunit

Polyamine Spider Toxins and Mammalian N‐Methyl‐d‐Aspartate Receptors

Desensitization of the P2X2 receptor controlled by alternative splicing

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Desensitization of the P2X₂ receptor controlled by alternative splicing