[19] Electrophysiological recording from Xenopus oocytes

doi:10.1016/0076-6879(92)07021-F

Methods in Enzymology

Volume 207, 1992, Pages 319-339

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This chapter describes various methods for electrophysiological measurements for studying for ion channels and transport systems in Xenopus oocytes. For most electrophysiological recordings from oocytes, the membrane potential has to be under control, and this is achieved by clamping the oocyte to predefined potentials using a conventional two-electrode voltage clamp. In most cases, problems in recording currents from oocytes damped with two electrodes arise from the electrodes themselves. Therefore, the possibility of measuring the electrode resistance during the experiment is very helpful. Any increase in electrode resistance deteriorates the clamp performance. The distribution of channels is not uniform over the membrane of the oocyte, and the magnitude of variation of the channel densities depends on the channel type. The two-electrode clamp of oocytes offers a series of advantages over patch clamp recording from macropatches—it is simpler, more stable, allows recording at lower channel densities, and the extracellular solution is easily changed.

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Citation Excerpt :
Xenopus oocytes are particularly advantageous because they only express small (100–200 pA) endogenous outward currents in the physiological range of membrane potentials. Detailed procedures for the use of Xenopus oocytes to study ion channel function have been previously described (Stühmer, 1992, 1998). In this section, we focus on specific procedures we have applied to study general anesthetic action on Kv channels, focusing on K-Shaw and Kv1.2.
Voltage-gated ion channels (VGICs) of excitable tissues are emerging as targets likely involved in both the therapeutic and toxic effects of inhaled and intravenous general anesthetics. Whereas sevoflurane and propofol inhibit voltage-gated Na⁺ channels (Navs), sevoflurane potentiates certain voltage-gated K⁺ channels (Kvs). The combination of these effects would dampen neural excitability and, therefore, might contribute to the clinical endpoints of general anesthesia. As the body of work regarding the interaction of general anesthetics with VGICs continues to grow, a multidisciplinary approach involving functional, biochemical, structural, and computational techniques, many of which are detailed in other chapters, has increasingly become necessary to solve the molecular mechanism of general anesthetic action on VGICs. Here, we focus on electrophysiological and modeling approaches and methodologies to describe how our work has elucidated the biophysical basis of the inhibition Navs by propofol and the potentiation of Kvs by sevoflurane.
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General anesthetics are known to act in part by binding to and altering the function of pentameric ligand-gated ion channels such as nicotinic acetylcholine and γ-aminobutyric acid type A receptors. Combining heterologous expression of the subunits that assemble to form these ion channels, mutagenesis techniques and voltage-clamp electrophysiology have enabled a variety of “structure–function” approaches to questions of where anesthetic binds to these ion channels and how they enhance or inhibit channel function. Here, we review the evolution of concepts and experimental strategies during the last three decades, since molecular biological and electrophysiological tools became widely used. Topics covered include: (1) structural models as interpretive frameworks, (2) various electrophysiological approaches and their limitations, (3) Monod–Wyman–Changeux allosteric models as functional frameworks, (4) structural strategies including chimeras and point mutations, and (5) methods based on cysteine substitution and covalent modification. We discuss in particular depth the experimental design considerations for substituted cysteine modification–protection studies.
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Ca^2 + concentration jumps for the activation of Ca^2 +-dependent ion channels or transporters can be obtained either by fast solution exchange or by the use of caged Ca^2 +. Here, we report on an alternate optogenetic method for the activation of Ca^2 + and voltage-dependent large conductance (BK) potassium channels. This was achieved through the use of the light-gated channelrhodopsin 2 variant, CatCh(Calcium translocating Channelrhodopsin) with enhanced Ca, which produces locally [Ca^2 +] in the μM range on the inner side of the membrane, without significant [Ca^2 +] increase in the cytosol. BK channel subunits α and β1 were expressed together with CatCh in HEK293 cells, and voltage and Ca^2 + dependence were analyzed. Light activation of endogenous BK channels under native conditions in astrocytes and glioma cells was also investigated. Additionally, BK channels were used as sensors for the calibration of the [Ca^2 +] on the inner surface of the cell membrane.
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Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na⁺]_i (e.g. during ischemia). An intracellular Na⁺ coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na⁺ coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na⁺ sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 m NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]_i of 70 mm. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na⁺. In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mm niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]_i from 3 to 100 mm. Thus, relative to fenamates, intracellular Na⁺ is a poor activator of Slo2.1.
Background: Molecular determinants for activation of Slo2.1 K⁺ channels by intracellular Na⁺ have not been identified.
Results: Charge reversal of Asp⁷⁵⁷ of Slo2.1 prevented activation of channels by intracellular Na⁺, whereas activation by niflumic acid was unaffected.
Conclusion: Asp⁷⁵⁷ is the primary residue responsible for sensing [Na⁺]_i in Slo2.1.
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Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.

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[19] Electrophysiological recording from Xenopus oocytes

Publisher Summary

Curr. Top. Dev. Biol.

Neuron

Nature (London)

Nature (London)

Nature (London)

Nature (London)

Nature (London)

Nature (London)

Nature (London)

Pfluegers Arch.