A PDZ-3 mediated physical and functional interaction between the CaV3.2 T-type calcium channel and neuronal nitric oxide synthase

Abstract

T-type voltage-gated calcium channels are expressed throughout the central and peripheral nervous systems as well as in several non-neuronal tissues and contribute to variety of functions such as neuronal excitability, intracellular calcium influx, shaping action potentials, pace-making activity, hormone secretion, and neurotransmitter release. Of the three T-type channel isoforms, Cav3.2 is uniquely sensitive to redox modulation with oxidizing reagents inhibiting and reducing compounds enhancing channel activity. This modulation has been shown to alter firing patterns of reticular thalamic neurons and to affect the nociceptive threshold in vivo suggesting that redox modulation of Cav3.2 may play an important role in regulating neuronal activity. A potential source of oxidizing molecules in vivo is neuronal nitric oxide synthase (nNOS), a calcium dependent enzyme which synthesizes nitric oxide (NO) from arginine. Interestingly, the carboxyl terminus of Cav3.2 possesses a putative PDZ-3 binding ligand which is compatible with the PDZ-3 domain of nNOS. I hypothesize that Cav3.2 and nNOS physically interact via the PDZ-3 binding ligand of Cav3.2 and that this physical interaction mediates a functional interaction whereby Cav3.2 activity stimulates nNOS to produce NO which, in turn, inhibits Cav3.2 activity. Cav3.2 and nNOS were expressed in a heterologous system which allowed us to examine the putative PDZ-3 mediated interactions between the two proteins. Immunoprecipitation experiments using Cav3.2 specific antibodies demonstrate that Cav3.2 and nNOS can interact via the carboxyl PDZ-3 ligand of Cav3.2 and that this interaction is disrupted when the PDZ-3 ligand is mutated. Utilizing a NO sensitive fluorometric assay we show that Cav3.2 activity can stimulate nNOS to produce NO and that disruption the PDZ-3 interaction precludes nNOS activation. We also demonstrate that the PDZ-3 mediated physical interaction facilitates the inhibition of Cav3.2 by nNOS derived NO. Disruption of the Cav3.2/nNOS interaction in vivo using intraperitoneal injection of membrane permeable peptides designed to competitively disrupt the PDZ-3 interaction produces an exaggerated respiratory response to changes in available oxygen and a blunted response in the hyperoxic response test. These results indicate that Cav3.2 and nNOS physically and functionally interact to contribute to normal physiological processes.