Rousset M et al. (2022), Mammalian Brain Ca2+ Channel Activity Transplan...

XB-ART-59125

Membranes (Basel) 2022 May 02;125:. doi: 10.3390/membranes12050496.

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Mammalian Brain Ca2+ Channel Activity Transplanted into Xenopus laevis Oocytes.

Rousset M , Humez S , Laurent C , Buée L , Blum D , Cens T , Vignes M , Charnet P .

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Several mutations on neuronal voltage-gated Ca2+ channels (VGCC) have been shown to cause neurological disorders and contribute to the initiation of epileptic seizures, migraines, or cerebellar degeneration. Analysis of the functional consequences of these mutations mainly uses heterologously expressed mutated channels or transgenic mice which mimic these pathologies, since direct electrophysiological approaches on brain samples are not easily feasible. We demonstrate that mammalian voltage-gated Ca2+ channels from membrane preparation can be microtransplanted into Xenopus oocytes and can conserve their activity. This method, originally described to study the alteration of GABA receptors in human brain samples, allows the recording of the activity of membrane receptors and channels with their native post-translational processing, membrane environment, and regulatory subunits. The use of hippocampal, cerebellar, or cardiac membrane preparation displayed different efficacy for transplanted Ca2+ channel activity. This technique, now extended to the recording of Ca2+ channel activity, may therefore be useful in order to analyze the calcium signature of membrane preparations from unfixed human brain samples or normal and transgenic mice.

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Species referenced: Xenopus laevis

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	Figure 1. Kinetic of receptor expression after injection of membrane preparation. (A) Typical current traces recorded in response to perfusion of GABA (1 mM) of control oocytes (N.I.) or oocytes injected with hippocampal brain membrane preparation (Hip), 6 h after injection (D0). (B) Kinetics of the response to GABA of oocytes injected with hippocampal brain membrane preparation quantified as the peak current amplitude recorded in response to GABA application (1 mM, HP = −80 mV, EC50 = 81µM) at D0: 6 h after membrane injection, and D1 and D2: 24 and 48 h after membrane injection, respectively. (C) Effect of injected hippocampal membrane concentration on the amplitude of the response of the oocytes to the perfusion of GABA. Two concentrations were used (3.5 and 7 mg/mL) and two time points were analyzed (D0 and D1).
	Figure 2. Expression of voltage-gated Ca2+ channels after injection of membrane preparation. (A) Typical current traces recorded on oocytes injected with water (H2O), or hippocampal membrane preparation in response to depolarizations to +20 mV from a holding potential of −80 mV, 5 h (5 H) or 24 h (24 H) after injection. The recording medium contained 40 mM Ba2+. (B) Expression was at its maximum level one day after injection (D1, set at 100%), and the current amplitude was similar at day 2 (D2) following membrane injection. (C) Histogram showing the ratio of the level of Ca2+ channel expression (measured as the peak current amplitude recorded during a depolarizing pulse to +10 mV in BANT40) on GABA receptor expression (measured as the peak current amplitude in response to 1 mM GABA at HP of −80 mV in ND86) for two different mice, M1 and M2. (D) Different membrane preparations (at 5 mg/mL) produced different levels of voltage-gated Ca2+ channel expression (N.I: non-injected oocytes, or oocytes injected with Hip: hippocampal membrane, Cerb: cerebellum membrane or Card: cardiac ventricular membrane). ns means—not significant, *—with injection.
	Figure 3. Biophysical properties of expressed voltage-gated Ca2+ channels. (A) Typical current traces recorded from an oocyte one day after injection of a hippocampal membrane preparation (7 mg/mL) during a twostep protocol. The first conditioning step had a duration of 2.5 s, and an amplitude varying from −80 to +40 mV in 10 mV increments, while the second step had a duration of 400 ms and remained at the same value ( +10 mV). Only some steps are displayed here. (B) Current–voltage curves obtained from similar protocols by plotting the relative peak current amplitude recorded during the first step as a function of the voltage of this step. These curves were obtained on oocytes injected with either hippocampal (Hip) or cerebellum (Cerb) membrane preparations, and Vact, kact, and Erev were 4.7 ± 0.4 mV; 7.7 ± 0.3 mV, 67 ± 1 mV (n = 8), and 6.8 ± 1.3 mV; and 8.4 ± 0.5 mV and 54 ± 2 mV (n = 11) for hippocampal and cerebellar membranes, respectively. (C) Isochronal inactivation curves obtained by plotting the relative peak current amplitude recorded during the second step as a function of the voltage of the first step. These curves were obtained on oocytes injected either with hippocampal (Hip) or cerebellum (Cerb) membrane preparations, and Vin, kin, and Rin were −25 ± 3 mV; 13 ± 1 mV, 0.1 ± 0.02 (n = 5), and −31 ± 4 mV; 15 ± 1 mV and 0.2 ± 0.01 (n = 10) for hippocampal and cerebellar membranes, respectively.
	Figure 4. Pharmacology of the transplanted voltage-gated Ca2+ channels. (A) Typical response of a hippocampal membrane-injected oocyte to 10 µM nifedipine. Voltage-gated Ca2+ currents were recorded during 400 ms-long depolarizations from −80 mV to +20 mV in the presence of 40 mM Ba2+. Right: histogram showing the average response to nifedipine. (B) Response of transplanted voltage-gated Ca2+ channels to the µ-opioid agonist DAMGO (10 µM). Oocytes were injected with hippocampal membrane preparation, and were re-injected 24 h later with either H2O (Hip) or the µ1 opiod receptor RNA at 1 ug/uL (Hip + µOR1). Recordings were made 48 h after the hippocampal membrane injection. (C) Average response to 10 µM DAMGO of transplanted voltage-gated Ca2+ channels co-injected or not with the µ1 opioid receptor RNA (D). Western blot using either hippocampal (Hip) or cerebellum (Cer) membrane preparations and probed with an anti-CaVβcom antibody showed that, in both cases, the cytoplasmic voltage-gated Ca channel auxiliary subunits, CaVβ, remained associated with the channel subunit(s). *—with injection.

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