Cyclic GMP-gated channels in a sympathetic neuron cell line

Abstract

The stimulation of IP3 production by muscarinic agonists causes both intracellular Ca2+ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3',5'-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide-gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na+ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na+ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na+ is 47 pS and is independent of voltage in the range -50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent KD of 10 microm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 microm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein-coupled neurotransmitter receptors can directly modify Ca2+ influx and electrical excitability. In N1E-115 cells, Ca2+ entry by this pathway is necessary to refill the IP3-sensitive intracellular Ca2+ pool during repeated stimulation and CNG channels may play a similar role in other neurons.

Figure 1

The membrane permeable cGMP analogue, 8-br-cGMP, activates cation channels in cell attached patches in differentiated N1E-115 cells. (A) Current records taken from a continuous recording before and 1, 3, 5, and 7 min after adding 1 mM 8-br-cGMP in the external saline. The pipette contained 153 mM NaCl, 10 mM EGTA, 1 mM EDTA, 1 μM TTX, and 10 HEPES (pH 7.4; 30°C). The pipette voltage was 0 mV and inward Na²⁺ currents are shown as downward deflections. Filter corner frequency = 1.5 kHz. (B) Amplitude histograms generated during 10-s recordings taken before and 1, 3, 5, 7, 11, and 14 min after adding 1 mM 8-br-cGMP (bin width 0.031pA). (C) Increase in the probability of one or more channels being open (NP_o) as a function of time before and after adding 1 mM 8-br-cGMP. NP_o was estimated by dividing the mean open time by the mean closed time during 10-s records taken at the times indicated. A 50% criterion was used to identify openings.

Figure 2

Cyclic GMP activates single channel currents in excised, inside out membrane patches. (A) Patch current during exposure to 10 μM cGMP for the period indicated by the horizontal bar. Both sides of the membrane were exposed to the same Na⁺ saline (composition in mM; 160 NaCl, 10 EGTA, 1 EDTA, and 10 HEPES, pH 7.4) and the potential across the patch was 40 mV, pipette positive. A dashed line shows the zero current level (filter cut-off frequency = 200 Hz). (B) A segment of the record is shown on an expanded time base to illustrate the burst structure (filter cutoff frequency = 1.5 kHz; sampled at 100 μs/pt). (C) Current amplitude histogram during exposure to 10 μM cGMP (filter cut-off frequency 1.5 kHz; bin width 0.03 pA). The distribution was fitted by the sum of two gaussian functions with mean amplitudes of zero and −1.44 pA using the method of least squares. The opening probability in 10 μM cGMP was 0.43.

Figure 3

(A) Current-voltage relationship for single channel currents in symmetrical Na⁺ solutions. Measurements were taken from the same patch during exposure to 10 μM cGMP applied to the inner membrane face. Points represent the mean current amplitude (±SD) for 10–12 well defined single openings at each voltage (filter cut-off frequency = 1.5 kHz). The solid line was fitted to the data by least-squares linear regression. (B) Open and closed time histograms of channel activity in response to 10 μM cGMP (0.3 ms bin size). The 20-s record used in this example contained 2431 events (filter cut-off frequency 1.5 kHz; 100 μs/pt.). The open time histogram was fitted by the sum of two exponentials (solid line) with time constants of 0.57 and 4.9 ms, accounting for 40 and 51% of all openings, respectively. The opening probability (P_o) calculated from the all points histogram was 0.46.

Figure 4

Effect of increasing cGMP concentration on channel activity. (A) Sample records from an inside out patch in symmetrical Na⁺ solutions. Solutions containing different concentrations of cGMP were applied to the inner face of the membrane by a multichannel perfusion device and channel activity is shown at four cGMP concentrations. The zero current level is indicated by dashed lines (pipette voltage = 40 mV; analog filter cutoff frequency = 500 Hz). (B) Amplitude histograms taken at four cGMP concentrations (0.026 pA/bin). The individual histograms were made from 10-s record segments (filter frequency = 1.5 kHz). The amplitude distribution with 100 μM cGMP was fitted by the sum of two gaussian functions and the fitted curve is shown by a smooth line superimposed on the data. The unitary current amplitude was −1.5 ± 0.2 pA (mean ± S.D.; 40 mV, pipette positive). P_o at 100 μM cGMP was 0.84.

Figure 5

Dose-response curve for single channel currents. Opening probability (P_o) is plotted as a function of cGMP concentration. Data were obtained by sampling 2-s to 2-min long record segments taken at four different cGMP concentrations (filter cutoff frequency; 1 kHz). Records with long closed periods were excluded from the analysis. Amplitude histograms were made from each record and fitted by the sum of two gaussian distributions. P_o was obtained either from the areas under the fitted curves or from measures of mean closed and mean open time using a 50% amplitude criterion. The points represent the mean (±S.D.) value of P_o from five single channel patches. The solid line is the nonlinear least-squares fit to the Hill equation (P = P _max · [cGMP/K _D]ⁿ/(1 + [cGMP/K _D]ⁿ)) using a dissociation constant, K _D, of 9.4 μM and Hill coefficient, n, of 2, assuming P _max = 1.

Figure 6

Sensitivity to cGMP and cAMP measured in the same single channel patch. A membrane patch was excised in symmetrical Na⁺ saline. The solution perfusing the inside face was changed every 30 s, switching between control saline and salines containing cGMP or cAMP at 50 and 100 μM concentrations. Records were taken 20 s after changing the solution flowing in the delivery pipette. Channel opening probability was 0.47 in 50 μM cGMP and 0.7 in 100 μM cGMP. cAMP failed to activate channel activity (filter corner frequency 100 Hz; 50 mV pipette potential; zero current level shown by dashed lines).

Figure 7

Evidence for multiple open and closed states. (A) Inside-out patch recording in symmetrical Na⁺ saline during continuous application of 100 μM cGMP to the inner face (filter corner frequency 1.5 kHz; 40 mV pipette potential). The patch contained a single CNG channel. The zero current level is shown by a dashed line. (B) Open time distribution (1 ms bin size). The sum of two exponential relaxations was fitted to the data by the method of least squares (time constants, 5 and 32 ms). (C) Closed time distribution. The square root of frequency is plotted against the log of time to emphasize longer closures. Greater than 85% of closures are less than 10 ms in duration in this example. (D) Amplitude histogram from the same experiment. The distribution was fitted by the sum of three gaussian functions with peaks at 0.0, −0.29, and −1.55 pA (solid line).

Figure 8

Evidence for a subconductance level. (A) Single channel currents in an inside out patch in symmetrical Na⁺ during stimulation with 100 μM cGMP. The zero current level is shown by the dashed line labeled closed and the maximum current is shown by the line labeled open. This figure illustrates a particularly long lasting transition to a level about 1/3 the maximum current amplitude (labeled sub). (B) Amplitude histogram generated from a 3-s record from the same experiment (bin width = 0.026 pA; filter corner frequency = 500 Hz). The sum of three gaussian functions was fitted to the data by the method of least squares (continuous line). From the areas under the fitted curves the probabilities of being in the closed state, the subconductance state, and the open state were 0.008, 0.05, and 0.93, respectively.