scholarly journals Identification of Cav2–PKCβ and Cav2–NOS1 complexes as entities for ultrafast electrochemical coupling

2017 ◽  
Vol 114 (22) ◽  
pp. 5707-5712 ◽  
Author(s):  
Cristina E. Constantin ◽  
Catrin S. Müller ◽  
Michael G. Leitner ◽  
Wolfgang Bildl ◽  
Uwe Schulte ◽  
...  

Voltage-activated calcium (Cav) channels couple intracellular signaling pathways to membrane potential by providing Ca2+ ions as second messengers at sufficiently high concentrations to modulate effector proteins located in the intimate vicinity of those channels. Here we show that protein kinase Cβ (PKCβ) and brain nitric oxide synthase (NOS1), both identified by proteomic analysis as constituents of the protein nano-environment of Cav2 channels in the brain, directly coassemble with Cav2.2 channels upon heterologous coexpression. Within Cav2.2–PKCβ and Cav2.2–NOS1 complexes voltage-triggered Ca2+ influx through the Cav channels reliably initiates enzymatic activity within milliseconds. Using BKCa channels as target sensors for nitric oxide and protein phosphorylation together with high concentrations of Ca2+ buffers showed that the complex-mediated Ca2+ signaling occurs in local signaling domains at the plasma membrane. Our results establish Cav2–enzyme complexes as molecular entities for fast electrochemical coupling that reliably convert brief membrane depolarization into precisely timed intracellular signaling events in the mammalian brain.

Author(s):  
Toshio Nakaki ◽  
Futoshi Shintani ◽  
Shigenobu Kanba ◽  
Eiji Suzuki ◽  
Masahiro Asai

1994 ◽  
Vol 304 (3) ◽  
pp. 683-686 ◽  
Author(s):  
C Harteneck ◽  
P Klatt ◽  
K Schmidt ◽  
B Mayer

Rat brain nitric oxide synthase was expressed to a high level in baculovirus-infected insect cells and purified to apparent homogeneity by affinity chromatography. The enzyme had a specific activity of approximately 1 mumol of citrulline.min-1.mg of protein-1 and contained 0.93, 0.45, 0.18 and 0.23 mol of haem, (6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin), FAD and FMN per mol of subunit respectively.


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