TRPC3 Knockout Cell Line (HEK293)

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSC) followed by a slow EPSC (EPSC). The latter is companied with a rise of intracellular Ca and critical for motor coordination. The genesis of EPSC in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSC. Electrophysiological analyses showed that EPSC in cerebellar slices of nNOS knockout (nNOS) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS mice evoked larger TRPC3-channel mediated currents and intracellular Ca rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSC, at least in part, by S-nitrosylation of STIM1.