Dynamin 2 is a Ca2+‐dependent regulator of NOS1α and a possible negative regulator of NOS1β

Abstract

Nitric oxide synthase 1 (NOS1)/NO pathway in the inner medullary collecting duct (IMCD) promotes natriuresis. Rat IMCD express NOS1α and NOS1β splice variants, while mouse IMCD express NOS1β. We reported dynamin 2 (DNM2) activates NO production via direct interaction with NOS1α. We hypothesized that 1) intracellular Ca2+ regulates DNM2/NOS1 interaction, and 2) knocking down or inhibiting DNM2 decreases NO production in IMCD. Reducing intracellular Ca2+ with BAPTA decreased the NOS1α/DNM2 interaction as determined by immunoprecipitation in NOS1α + DNM2 transfected COS7 cells. While increasing intracellular Ca2+ with ionomycin, enhanced the NOS1α/DNM2 interaction. Thus, NOS1α/DNM2 interactions are Ca2+‐dependent. We confirmed that NOS1β also interacts with DNM2 in mouse IMCD3 cells. NO production, assessed by HPLC, was similar in control and DNM2 siRNA (95% knockdown confirmed) groups (P>;0.05). NO production was significantly increased with DNM2 inhibitors (ranged from 1410 ± 419 to 1910±105 pmol/mg pr/h) compared to controls (ranged from 665 ± 57 to 839 ± 120 pmol/mg pr/h; n = 4, P < 0.05). Reducing expression or inhibition of DNM2 in mouse IMCD, which express NOS1β only, either has no effect or increases NO production. These data suggest that DNM2 acts as a Ca2+‐dependent positive regulator of NOS1α and a possible negative effector of NOS1β. Funding support: APS to SKHM; AHA to KAH; NIH to JSP.