Biomedical Chemistry: Research and Methods, 2018, 1(3), e00034
The 40th Anniversary of the Institute of Physiologically Active Compounds of the Russian Academy of Sciences

Properties of Calcium-Activated Chloride Currents in Rat Purkinje Cerebellum Neurons

V.L. Zamoyski*, E.V. Bovina, V.V. Grigoriev

Institute of Physiologically Active Compounds of the Russian Academy of Sciences, 1 Severny proezd, Moscow region, Chernogolovka, 142432 Russia;*e-mail: vzam@yandex.ru

Key words: patch-clamp method; Purkinje cerebellum cells; calcium-activated chloride current; tetraethylammonium; 4-aminopyridin; nifuminic acid

DOI: 10.18097/BMCRM00034

The whole version of this paper is available in Russian.

The presence of calcium-activated chloride current was shown using on freshly isolated rat Purkinje cerebellum neiurones and the pacth-clamp method in the whole-cell configuration. Chloride currents appeared in sodium-free external solution and reversibly disappeared in chloride-free or calcium-free external solution. Replacing of K+ ions (120 mM) to Cs+ ions in micropipette (120 mM) show the chloride currents even with 140 mM Na+ in external solution. This current was blocked to 80–100% by nifluminic acid (25–100 μM). It was found out that well known blockers of potassium channels tetraethylammonium (TEA) and 4-aminopyridine (4-AP) also effectively blocked chloride channels. The IC50 values for TEA and 4-AP were 130 μM, and 110 μM respectively. The action of TEA was reversible, while 4-AP at concentration 100 μM and above irreversibly blocked chloride channels.

Figure 1. Currents in the whole-cell configuration in response to rectangular depolarization pulses with step +10 mV (at left side) and their Voltage-Current relationships (at right side). The micropipette was filled by solution B1 (see table 1), outside solution is H1.

Figure 2. Currents in the whole-cell configuration in response to rectangular depolarization pulses with step +10 mV (at left side) and their Voltage-Current relationships (at right side). The micropipette was filled by solution B1 (see table 1), outside solution without sodium is H2.

Figure 3. Currents in the whole-cell configuration in response to rectangular depolarization pulses with step +10 mV (at left side) and their Voltage-Current relationships (at right side). The micropipette was filled by solution B1 (see table 1), outside solution without sodium and chloride ions is H4.

Figure 4. Integral currents of the Purkinje neuron and their voltage-current relationships in the case of solution B2 in micropipettes (cesium).
a — Integral currents in response on series of rectangular depolarization pulses from –60 to +20 mV on cell membrane with the H1 (sodium) solution ouside of the cell membrane.
b — Voltage-currents relationship of currents shown on the fig. 2,a.
c — Integral currents in response on series of rectangular depolarization pulses from –60 to +20 mV on cell membrane with the H2 (sodium-free) solution ouside of the cell membrane.
d — Voltage-currents relationship of currents shown on the fig. 2,c.

Figure 5. The effect of nifluminic acid (NFA) on chloride currents in the Purkinje neuron. Protocol of rectangular depolarization pulses with step
+10 mV from holding potential –70 mV is shown at the top of the picture.
a — Ionic currents in response or pulses serial. Outside of the membrane solution H2 (sodium-free), solution B2 (cesium) in micropipette.
b — Ionic currents after adding of 100 μM NFA.
c — Wash out of NFA by solution H2 during 5 min.

Figure 6. The effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on chloride currents.
a — Response of the cell on the series of rectangular electric pulses (track 1) in control (track 2), during action of 1 mM of TEA (track 3), and after washout (track 4). During the TEA action outside cation current and outside chloride current were blocked to 17 and 20% respectively, at +20 mV on the membrane.
b — In the same (see Fig 4,a) conditions, the other cell, show the control response (track 2), during action of 100 uM of 4-AP (track 3), and response after washout of 4-AP. The blockadge of outside and chloride currants were 47% and 44%, respectively, at the potential +20 mV on cell membrane.

CLOSE
Table 1. Ionic composition of solutions.

ACKNOWLEDGEMENTS

The equipment of the Centre of collective usage of IPAS RAS was used in the work (Agreement No. 14.621.21.0008,
work identifier RFMEFI62114X0008).

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