Nitric Oxide Inhibits BK Currents In Cultured Hippocampal Neurons Via S-nitrosylation

Nitric oxide (NO) is a gas molecule with free radical properties and functions as a transmitter molecule in both the peripheral and central nervous systems. It is synthesized during the catalytic conversion of arginine to citrulline by the enzyme nitric oxide synthase (NOS). Diffusing freely, NO is involved in many physiological and pathological processes such as neurotransmitter release, long-term potentiation and depression, smooth-muscle relaxation, protein-protein interactions, proliferation and differentiation, apoptosis, excitotoxicity, redistribution of intracellular oxygen in hypoxia.Large conductance Ca²⁺-activated potassium channels (BK) are widely distributed in neurons, muscles, and gland cells and play a variety of roles in physiology, generally providing an inhibitory negative feedback influence that links cellular metabolism and excitability. Moreover, our recent studies indicate an enhancement of BK channel currents in hippocampus after ischemia. However, the underlying mechanisms are unclear. The regulation of BK channels by NO has been extensively studied in non-neuronal cells while there is no data available in the NO-mediated modulation of neuronal BK channels.In the present study, the effect and mechanism of nitric oxide (NO) on BK currents in cultured hippocampal neurons and CHO cells expressed hSlola subunit were studied by using whole-cell patch clamp recordings. L-arginine (L-ARG, 2 mM/L) depressed the whole outward K⁺ currents in cultured hippocampal neurons by 37.5 %, of which BK currents were depressed about by 20%, while D-arginine (D-ARG, 2 mM/L) exerted no effect. The results indicate that L-ARG exerted itsfunction by its metabolizing but not by itself. Next, we proved that it was NO that inhibited K+ currents. In the presence of a specific inhibitor of NOS (L-NAME, 0.5 mM/L), the depressing effect by L-ARG was completely blocked, indicating that the inhibition of L-ARG is due to NO production.Considerable evidences indicate that NO exerts action through activating guanylate cyclase (GC) and increasing levels of cGMP. In addition, accumlating studies show that NO modulates protein function through direct modification of their sulphhydryl groups, which is named nitrosylation.On the basis of these observations, we performed further experiments to test whether cGMP-dependent pathway is involved in the NO-mediated inhibition of the BK currents in hippocampal neurons. After pretreatment with IQmM/L ODQ, a specific inhibitor of GC, to block cGMP-dependent pathway, 2 mM/L L-ARG inhibited the whole outward K+ currents by 24.5%, which is no significant difference compared with 2 mM/L L-ARG alone group (37.5%). This result suggests that NO modulates K+ currents mainly through cGMP-independent pathways. In order to isolate BK currents from other K+ currents, we pretreated hippocampal neurons with IQmM/L paxilline and 10//M/L ODQ, and then observed the effect of L-ARG. The remained K+ currents were inhibited by 25.3% with L-ARG. There is no significant difference in the inhibition between pretreatment with paxilline and ODQ and with paxilline alone (one way ANOVA, p>0.05). These results suggest that NO inhibits BK currents and other outward K+ currents both mostly through cGMP-independent pathways.JV-ethylmaleimide (NEM) is known to covalently modify protein sulphhydryl groups and make them incapable of nitrosylation 8. Pretreatment with 1 mM/L NEM for 10 minutes completely precluded L-ARG-induced inhibition on the whole K+ currents, suggesting that NO modulates K+ currents mostly through nitrosylation.No BK current was recorded in wild type CHO cells by using inside-out excised patch clamp technique. However, we recorded typical macroscopic BK currents from those CHO cells successfully expressed hSlola subunit within 24-48h in the presence of 2/iM/L intracellular Ca2+. These currents were augmented by 27.4% with BK channel opener 10/iM/L NS1619. Application of lmM/L GSNO (an NO donor) to the cytoplasmic side in the inside-out configuration markedly decreased the currents by 35.2 % when given depolarization to +60 mV from a holding potential of -70mV. The result that NO is still able to inhibit BK currents in the cell-free excised patches in the absence of ATP and GTP further supports a cGMP-independent action on BKchannels.The present results of both whole-cell recordings in hippocampal neurons and cell-free recordings in patches expressed hSlola subunit are consistent with the hypothesis that NO-induced inhibition of BK currents is independent of cGMP signaling pathways. Our study also indicates that NO inhibits BK currents through S-nitrosylation.