xenopus-oocytes-optimized-methods-for-microinjection-removal

<em>Xenopus</em> Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

University of Bern

The present study examined the effects and mechanisms of actions of penta-m-digalloyl-glucose (PDG), a hydrolysable tannin extracted from Chinese gallnut, on cystic fibrosis transmembrane conductance regulator protein (CFTR).

A plant-derived hydrolysable tannin inhibits CFTR chloride channel: a potential treatment of diarrhea.

Olfactory cyclic nucleotide-gated (CNG) ion channels are essential contributors to signal transduction of olfactory sensory neurons. The activity of the channels is controlled by the cyclic nucleotides guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP). The olfactory CNG channels are composed of two CNGA2 subunits, one CNGA4 and one CNGB1b subunit, each containing a cyclic nucleotide-binding domain. Using patch-clamp fluorometry, we measured ligand binding and channel activation simultaneously and showed that cGMP activated olfactory CNG channels not only by binding to the two CNGA2 subunits but also by binding to the CNGA4 subunit. In a channel in which the CNGA2 subunits were compromised for ligand binding, cGMP binding to CNGA4 was sufficient to partly activate the channel. In contrast, in heterotetrameric channels, the CNGB1b subunit did not bind cGMP, but channels with this subunit showed activation by cAMP. Thus, the modulatory subunits participate actively in translating ligand binding to activation of heterotetrameric olfactory CNG channels and enable the channels to differentiate between cyclic nucleotides.

Differential regulation by cyclic nucleotides of the CNGA4 and CNGB1b subunits in olfactory cyclic nucleotide-gated channels.

In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.

Quantifying the cooperative subunit action in a multimeric membrane receptor.

GABAA receptors are pentameric ligand-gated channels mediating inhibitory neurotransmission in the CNS. α4βxδ GABAA receptors are extrasynaptic receptors important for tonic inhibition. The functional properties and subunit arrangement of these receptors are controversial. We predefined subunit arrangement by using subunit concatenation. α4, β2, and δ subunits were concatenated to dimeric, trimeric, and, in some cases, pentameric subunits. We constructed in total nine different receptor pentamers in at least two different ways and expressed them in Xenopus oocytes. The δ subunit was substituted in any of the five positions in the α1β2 receptor. In addition, we investigated all receptors with the 2:2:1 subunit stoichiometry for α4, β2, and δ. Several functional receptors were obtained. Interestingly, all of these receptors had very similar EC50 values for GABA in the presence of the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC). All functional receptors containing δ subunits were sensitive to 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide (DS2). Moreover, none of the receptors was affected by ethanol up to 30 mm These properties recapitulate those of non-concatenated receptors expressed from a cRNA ratio of 1:1:5 coding for α4, β2, and δ subunits. We conclude that the subunit arrangement of α4β2δ GABAA receptors is not strongly predefined but is mostly satisfying the 2:2:1 subunit stoichiometry for α4, β2, and δ subunits and that several subunit arrangements result in receptors with similar functional properties tuned to physiological conditions.

Toward Understanding Functional Properties and Subunit Arrangement of α4β2δ γ-Aminobutyric Acid, Type A (GABAA) Receptors.

Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.

Nisa Wongsamitkul

Institute of Biochemistry and Molecular Medicine

University of Bern

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Nisa Wongsamitkul

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Biochemistry and Molecular Medicine

University of Bern

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Institute of Biochemistry and Molecular Medicine