Hydrolysis of PIP2 is Responsible for the Voltage-Independent Inhibition of Cav2.2 Channels

Abstract

GPCRs regulate CaV2.2 channels through both voltage-dependent and -independent inhibition pathways. Gβγ subunits are the effectors of the voltage-dependent inhibition while the molecular mechanism for the voltage-independent inhibition remains to be elucidated. Here, we addressed whether the hydrolysis of PIP2 is responsible for the inhibition remaining after a conditioning pulse, named as voltage-independent inhibition. We recorded CaV2.2 currents from rat superior cervical ganglion neurons. We isolated the voltage-independent inhibition with a double-pulse protocol and assumed that the inhibition remaining after a conditioning pulse corresponds to the voltage-independent pathway. To test our hypothesis, we changed the PIP2 concentration by means of blocking phospholipase C, filling the cell with a PIP2 analogue, and preventing PIP2 re-synthesis. The internal dialysis of short-chain PIP2 was firstly assessed during the muscarinic inhibition of KCNQ2/3 channels. First, we determined the required magnitude, duration, and interval between the conditioning and second pulses to isolate the voltage-independent inhibition. Our results show that the inhibition of phospholipase C reduces the inhibition remaining after a conditioning pulse upon noradrenaline or muscarinic application. Dialyzing into the cell micromolar concentration of short-chain PIP2, through the recording pipette, hampers the muscarinic inhibition of KCNQ2/3 current in tsA-201 cells as well as the voltage independent inhibition of CaV2.2 channels in neurons. Also, blocking the phosphatidylinositol 4-kinase with wortmannin avoids the recovery from the voltage independent inhibition. These results support the hypothesis that the hydrolysis of PIP2 is responsible of the voltage-independent inhibition of CaV2.2. Regulation of levels of PIP2 in nerve terminals can result in the regulation of presynaptic calcium channels and in the subsequent modulation of exocytosis.This work was supported by UNAM- DGAPA-PAPIIT (IN200710).