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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
77
35-POS
Board 35
Structural and Functional Relations of the Voltage-dependent Gated ClC-2 Chloride
Channel Assessed by Voltage Clamp Fluorometry.
Irma L. González-Hernández
, Roberto Gastélum-Garibaldi, Marina S. Rodríguez-Rangel,
Denise M. Alonso-Vázquez, Jorge E. Sánchez-Rodríguez.
Universidad de Guadalajara, Guadalajara, Mexico.
Voltage clamp fluorometry (VCF) is a powerful technique that provides simultaneous
information on the function and conformation changes of an ion channel protein with a high
temporal resolution. Here, we used VCF to gain further insights into conformational
rearrangements that the voltage-dependent gated ClC-2 chloride (Cl-) channel experiments
during its function. To this end, we engineered single cysteine mutants on the external facing of
the P and Q α-helices of the mouse ClC-2 chloride channel as targets of a cysteine-reactive
fluorescent reporter (TMR). Wild type ClC-2 channel and all cysteine mutants were expressed in
Xenopus laevis oocytes. Its functional integrity, pH and Cl- concentration dependence were
assessed by using the cut-open oocyte voltage clamp technique. In agreement with the
homodimeric architecture ClC chloride channels, the ionic currents produced by the ClC-2
channels displayed a well-defined time dependence of the fast gating and slow/common gating.
Importantly, we have identified the position Q535C (top Q helix) that produce voltage dependent
fluorescence changes during its opening and closing gating. These fluorescence intensity changes
from Q535C were correlated with its respective open probability curve following a Boltzmann
distribution. This finding begins to define an external region of the ClC-2 that is moving during
its opening and closing transitions. Suported by PROSNI-UDG/2017 and PRODEP-NPTC
236463 to JESR.