CYCLIC GMP-GATED CHANNELS IN A SYMPATHETIC NEURON CELL-LINE

The stimulation of IP3 production by muscarinic agonists causes both i ntracellular Ca2+ release and activation of a voltage-independent cati on current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the m embrane current requires an increase in 3',5'-cyclic guanosine monopho sphate (cGMP) produced through the NO-syntase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide-gated ion channels. This was tested using patch clamp methods. The membrane perm eable cGMP analogue, 8-br-cGMP, activates Na+ permeable channels in ce ll attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na+ solutions. cGMP-dependent single cha nnel activity consists of prolonged bursts of rapid openings and closi ngs that continue without desensitization. The rate of occurrence of b ursts as well as tile bur-st length increase with cGMP concentration. The unitary, conductance in symmetrical 160 mM Na+ is 47 pS and is ind ependent of voltage in the range -50 to +50 mV. There is no apparent e ffect of voltage on opening probability. The dose response curve relat ing cGMP concentration to channel opening probability is fit by the Hi ll equation assuming an apparent K-D of 10 mu m and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrati ons as high as 100 mu m Cyclic nucleotide gated (CNG) channels in N1E- 115 cells share a number of properties with CNG channels in sensory re ceptors. Their presence in neuronal cells provides a mechanism by whic h activation of the NO/cGMP pathway by G-protein-coupled neurotransmit ter receptors can directly modify Ca2+ influx and electrical excitabil ity. In N1E-115 cells, Ca2+ entry by this pathway is necessary to refi ll the IP3-sensitive intracellular Ca2+ pool during repeated stimulati on and CNG channels may play a similar role in other neurons.