Xenopus Oocyte Microinjection and Ion-Channel Expression

Smart, TG; Krishek, Belinda J.

doi:10.1385/0-89603-311-2:259

Cited by 16 publications

(12 citation statements)

References 57 publications

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“…Oocytes were removed from anaesthetized Xenopus laevis following immersion in 0.5% tricaine as described previously (Smart & Krishek, 1995) and stored in modified Earth's medium (MBM) containing (m m ): 110 NaCl, 1 KCl, 2.4 NaHCO 3 , 7.5 Tris‐HCl, 0.33 Ca(NO 3 ) 2 , 0.41 CaCl 2 , 0.82 MgSO 4 , and 50 μg ml −1 gentamicin; pH 7.6. Stage IV and V oocytes were separated and centrifuged (700–1100 g for 7–8 min at 10 °C) for nuclear microinjection with 10 nl of 1 mg ml −1 DNA solution, encoding either for the murine β3, β3H292A, αlβ3 (ratio of 1:1) or αlβ3H292A (1:1) GABA A receptor subunits.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Zn²⁺ binding site on the marine gABA_A receptor complex: Dependence on the Second transmembrane domain of β subunits

Wooltorton

McDonald

Moss

et al. 1997

The Journal of Physiology

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Whole‐cell currents were recorded from Xenopus laevis oocytes expressing wild‐type and mutant recombinant GABAA receptors to locate a binding site for Zn2+ ions in the β3 subunit. The Cl−‐selective current, spontaneously gated by β3 subunit homomers, was enhanced by pentobarbitone and inhibited by picrotoxinin. The potencies of these agents were minimally affected by mutating histidine (H) 292 to alanine (A) in the second transmembrane domain (TM2). Zn2+ inhibited the β3 subunit‐gated conductance (IC50, 0.31 μm); the inhibition was voltage insensitive. The H292A mutation in β3 subunits caused a 1000‐fold reduction in Zn2+ potency (IC50, 307 μm). GABA‐activated responses recorded from heteromeric α1β3 GABAA receptors were also inhibited by Zn2+ (IC50, 0.11 μm). This inhibition was reduced by mutating H292A in the β3 subunit (IC50, 22.8 μm). H292 in TM2 of the β3 subunit is an important determinant of a Zn2+ binding site on the GABAA receptor. Its location in the presumed ion channel lining suggests that Zn2+ can penetrate into an anion‐selective channel and that the ionic selectivity filter and channel gate are located beyond H292.

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Section: Methodsmentioning

confidence: 99%

Identification of a Zn²⁺ binding site on the marine gABA_A receptor complex: Dependence on the Second transmembrane domain of β subunits

Wooltorton

McDonald

Moss

et al. 1997

The Journal of Physiology

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“…Stage IV-V Xenopus laevis oocytes were obtained using a method similar to that previously reported (Smart and Krishek. 1995).…”

Section: Cell Preparation: Oocyte Extraction and Microinjectionmentioning

confidence: 99%

Pharmacological and Physiological Characterization of Murine Homomeric β3 GABA_A Receptors

Wooltorton

Moss

Smart

1997

Eur J of Neuroscience

119

122

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Gamma-Aminobutyric acid (GABA[A]) receptor beta3 subunits were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamp. Injected oocytes exhibited an increased resting membrane conductance and more depolarized membrane potentials compared to uninjected control cells. Oocytes expressing beta3 subunits were insensitive to GABA and muscimol, but pentobarbitone increased the membrane conductance in a concentration-dependent manner. The membrane current response to pentobarbitone reversed at the Cl- equilibrium potential and at relatively high concentrations (> 500 microM), a rebound Cl- current was induced following the removal of pentobarbitone. In transfected human embryonic kidney (HEK) cells, the rebound current amplitude was reduced by desensitizing the beta3 receptor with increased durations of ligand application. Both picrotoxin (0.5 nM to 10 microM) and Zn2+ (10 nM to 100 microM) reduced the resting membrane conductance for beta3 cDNA-injected oocytes. These oocytes were insensitive to flurazepam (5 microM) and alphaxalone (10 microM), but responded with increased membrane conductance to propofol (10 microM) and pregnanolone (50 nM to 5 microM). The antagonists, bicuculline (10 microM) and strychnine (50 nM to 100 microM), also induced conductance increases in a concentration dependent manner; however, glycine (1 mM) was inactive. It was concluded that beta3 subunits form spontaneously opening ion channels that can be up-regulated by some allosteric modulators, principally by pentobarbitone and propofol and, surprisingly, by bicuculline and strychnine, whilst picrotoxin and Zn2+ acted as antagonists. Computer modelling of some kinetic schemes was used to describe the rebound current observed in transfected HEK cells. This indicated that pentobarbitone, after modulation of the conductance, is potentially capable of further binding to the beta3 receptor complex 'driving' the receptor into one or more desensitized states. This phenomenon may be of some importance for native neuronal GABA(A) receptors, where pentobarbitone can also evoke rebound current activation.

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“…The Xenopus laevis oocyte is another classical cell of choice for electrophysiologists (20) but its opacity limits optical control and is therefore not recommended for studying light-regulation of ionic currents.…”

Section: Methodsmentioning

confidence: 99%

Photochromic Potassium Channel Blockers: Design and Electrophysiological Characterization

Mourot

Fehrentz

Krämer

2013

Methods in Molecular Biology

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Voltage-gated potassium (Kv) channels are membrane proteins that open a selective pore upon membrane depolarization, allowing K+ ions to flow down their electrochemical gradient. In neurons, Kv channels play a key role in repolarizing the membrane potential during the falling phase of the action potential, often resulting in an after hyperpolarization. Opening of Kv channels results in a decrease of cellular excitability, whereas closing (or pharmacological block) has the opposite effect, increased excitability. We have developed a series of photosensitive blockers for Kv channels that enable reversible, optical regulation of potassium ion flow. Such molecules can be used for remote control of neuronal excitability using light as an on/off switch. Here we describe the design and electrophysiological characterization of photochromic blockers of ion channels. Our focus is on Kv channels but in principle, the techniques described here can be applied to other ion channels and signaling proteins.

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Xenopus Oocyte Microinjection and Ion-Channel Expression

Cited by 16 publications

References 57 publications

Identification of a Zn²⁺ binding site on the marine gABA_A receptor complex: Dependence on the Second transmembrane domain of β subunits

Identification of a Zn²⁺ binding site on the marine gABA_A receptor complex: Dependence on the Second transmembrane domain of β subunits

Pharmacological and Physiological Characterization of Murine Homomeric β3 GABA_A Receptors

Photochromic Potassium Channel Blockers: Design and Electrophysiological Characterization

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Xenopus Oocyte Microinjection and Ion-Channel Expression

Cited by 16 publications

References 57 publications

Identification of a Zn2+ binding site on the marine gABAA receptor complex: Dependence on the Second transmembrane domain of β subunits

Identification of a Zn2+ binding site on the marine gABAA receptor complex: Dependence on the Second transmembrane domain of β subunits

Pharmacological and Physiological Characterization of Murine Homomeric β3 GABAA Receptors

Photochromic Potassium Channel Blockers: Design and Electrophysiological Characterization

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Identification of a Zn²⁺ binding site on the marine gABA_A receptor complex: Dependence on the Second transmembrane domain of β subunits

Identification of a Zn²⁺ binding site on the marine gABA_A receptor complex: Dependence on the Second transmembrane domain of β subunits

Pharmacological and Physiological Characterization of Murine Homomeric β3 GABA_A Receptors