Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

16-POS Board 16 Interacting PIP 2 Electrostatic Charges Account for Voltage-independent Regulation of CaV2.2 Calcium Channels Hector Castro , Karina Bermeo, Isabel Arenas, David Garcia. Universidad Nacional Autónoma de México, Mexico, Ciudad de Mexico, Mexico. Membrane lipids are key determinants in the regulation of voltage-gated ion channels. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ), a membrane phospholipid, has been studied in the regulation of voltage-gated calcium channels (VGCC). However, the nature of the voltage- independent regulation of VGCC has not been fully elucidated. The aim of this work was to investigate whether the interaction of PIP 2 electrostatic charges are responsible for the voltage- independent regulation of Ca V 2.2 channels. By using biophysical and biochemical methods, charge shielding of PIP 2 was performed in superior cervical ganglion (SCG) neurons of the rat. Firstly, we activated Gq/11 signaling by applying 10 µM oxotremorine-M (oxo-M), a muscarinic agonist, and measured the voltage-independent regulation by using a double-pulse protocol. We characterized the voltage-independent inhibition of the calcium current through the activation by oxo-M of a PIP 2 -mediated signaling pathway. To determine whether phospholipase C (PLC) activation was involved in this signaling cascade, cells were treated with U-73122, a PLCβ blocker. As expected, it reduced the muscarinic calcium current inhibition. Likewise, dialysis of 100 µM diC8-PIP 2 attenuated the muscarinic inhibition on calcium currents. Since PIP 2 hydrolysis is required to calcium current inhibition by oxo-M, we tested ATP-dependency on the recovery from muscarinic inhibition. After several oxo-M applications, we observed no differences in the current recovery by decreasing ATP concentrations, indicating that PIP 2 resynthesis is not involved. Finally, to test whether PIP 2 binds directly to the calcium channel, we used neomycin. This polycation has been shown to block electrostatic interactions of PIP 2 with some proteins, such as PLC and ion channels. Accordingly, neomycin reduced calcium current amplitude in a voltage-independent fashion. These data support that interacting PIP 2 charges underlies the voltage-independent regulation of calcium channels in SCG neurons. Supported by grants, PAPIIT: IN218016, IA206317, IV100116 and CONACyT: 255635.

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