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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
44
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.