Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

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.

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