Intracellular Na⁺ and metabolic modulation of Na/K pump and excitability in the rat suprachiasmatic nucleus neurons

Na/K pump activity and metabolic rate are both higher during the day in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigated the role of intracellular Na⁺ and energy metabolism in regulating Na/K pump activity and neuronal excitability. Removal of extracellular K⁺ to block the Na/K pump excited SCN neurons to fire at higher rates and return to normal K⁺ to reactivate the pump produced rebound hyperpolarization to inhibit firing. In the presence of tetrodotoxin to block the action potentials, both zero K⁺ -induced depolarization and rebound hyperpolarization were blocked by the cardiac glycoside strophanthidin. Ratiometric Na imaging with a Na⁺ -sensitive fluorescent dye indicated saturating accumulation of intracellular Na⁺ in response to pump blockade with zero K⁺. The Na⁺ ionophore monensin also induced Na⁺ loading and hyperpolarized the membrane potential, with the hyperpolarizing effect of monensin abolished in zero Na⁺ or by pump blockade. Conversely, Na⁺ depletion with Na⁺ -free pipette solution depolarized membrane potential but retained residual Na/K pump activity. Cyanide inhibition of oxidative phosphorylation blocked the Na/K pump to depolarize resting potential and increase spontaneous firing in most cells, and to raise intracellular Na⁺ levels in all cells. Nonetheless, the Na/K pump was incompletely blocked by cyanide but completely blocked by iodoacetate to inhibit glycolysis, indicating the involvement of both oxidative phosphorylation and glycolysis in fueling the Na/K pump. Together, the results indicate the importance of intracellular Na⁺ and energy metabolism in regulating Na/K pump activity as well as neuronal excitability in the SCN neurons.