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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
112
66-POS
Board 66
Gating the ClC-2 Chloride Channel by Permeant Anions
Jorge Sanchez Osuna
1
, Jorge Arreola Gómez
1
, Arlette Méndez Maldonado
2
,
1
Autonomous University of San Luis Potosí, San Luis Potosi, San Luis Potosí, Mexico,
2
Autonomous University of San Luis Potosí, San Luis Potosi, San Luis Potosí, Mexico.
The activation of the homodimeric ClC-2 Cl- channel is voltage dependent; however, the channel
lacks classical voltage-sensing domains. The closed to open transition is initiated by dislodging
the negatively charged side chain of an acid glutamic residue (E213), which constitutes the gate
located inside the pore. We hypothesize that a hyperpolarization introduces intracellular anions
into the pore and forces the gate to open to allow anion permeation. In the present work, we
analyze the gating mechanism of CLC-2 using experimental and computational
electrophysiology. We determine the effects of different concentrations of intracellular acetate
(0, 20, 60, 80 mM), a low permeant anion (Pace/PCl = 0.04), mixed with 60 mM Cl. In addition,
intracellular Cl concentrations (0, 20, 40, 60 mM) mixed with 80 mM acetate. Cells were bathed
in a solution containing 140 mM Cl- and voltage dependent activation was evaluated by
measuring the tail current at +60 mV after channel activation in the range of +60 to -200 mV.
We found that increasing the acetate concentration from 0 to 80 mM shifted channel activation
from -140.2 mV to -87.4 mV. In contrast, increasing the intracellular Cl from 0 to 60 mM shifted
the channel activation from -110.4 mV to -69.5 mV. We concluded that activation was mediated
by multi-ion occupancy and that the pore occupation by anion is a necessary step to open the
gate. To infer the molecular events leading to channel opening by the anion-gate interaction, we
used molecular dynamics simulation (computation electrophysiology). We embedded CLC-2
into a dipalmitoyl-phosphatidylcholine membrane and exposed to symmetrical 140 mM NaCl. A
voltage difference of -140 mV drive two Cl- ions from the intracellular side into the pore. The
leading Cl- interacts electrically with E213 side chain to unblock the conduction pathway.