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Cyclic GMP-gated channels in a sympathetic neuron cell line.

Thompson SH - J. Gen. Physiol. (1997)

Bottom Line: There is no apparent effect of voltage on opening probability.In contrast, cAMP failed to activate the channel at concentrations as high as 100 microm.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and the Hopkins Marine Station, Stanford University, Pacific Grove, California 93950, USA. stuartt@leland.stanford.edu

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.

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(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  (Po) calculated from the all points histogram was 0.46.
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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 (Po) calculated from the all points histogram was 0.46.

Mentions: Single channel currents were recorded from inside out patches exposed to symmetrical Na+ solutions. In the example in Fig. 2, the voltage across the patch was 40 mV, pipette positive, and the inside face of the membrane was perfused with saline containing 10 μM cGMP for the period indicated (Panel A). This rapidly and reversibly activated a Na+ permeable channel. Channel openings occurred in bursts separated by silent periods and only rarely as isolated single openings. There was no evidence of desensitization during exposures to cGMP lasting several minutes. A portion of the record is shown on an expanded time scale in Panel B to illustrate the burst structure. In the presence of cGMP, the channel flickers rapidly between open and closed states and some of these events are too brief to fully resolve. Panel C shows the distribution of patch current amplitudes during exposure to 10 μM cGMP. In this and each of the following examples the amplitude histogram was free of points that might indicate the presence of more than one channel. The solid line represents the sum of two gaussian distributions fitted to the data. The channel undergoes transitions from a closed state to an open state with a mean current amplitude of −1.44 pA at this voltage. In the presence of 10 μM cGMP the opening probability (Po) estimated from the areas under the fitted curves was 0.43. The I(V) curve for single channel currents was measured by varying the pipette potential and is linear in the range −50 to +60 mV in symmetrical Na+ solutions with an intercept near zero mV and a slope conductance of 47pS (Fig. 3 A). There was no obvious effect of pipette potential on opening probability.


Cyclic GMP-gated channels in a sympathetic neuron cell line.

Thompson SH - J. Gen. Physiol. (1997)

(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  (Po) calculated from the all points histogram was 0.46.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233783&req=5

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 (Po) calculated from the all points histogram was 0.46.
Mentions: Single channel currents were recorded from inside out patches exposed to symmetrical Na+ solutions. In the example in Fig. 2, the voltage across the patch was 40 mV, pipette positive, and the inside face of the membrane was perfused with saline containing 10 μM cGMP for the period indicated (Panel A). This rapidly and reversibly activated a Na+ permeable channel. Channel openings occurred in bursts separated by silent periods and only rarely as isolated single openings. There was no evidence of desensitization during exposures to cGMP lasting several minutes. A portion of the record is shown on an expanded time scale in Panel B to illustrate the burst structure. In the presence of cGMP, the channel flickers rapidly between open and closed states and some of these events are too brief to fully resolve. Panel C shows the distribution of patch current amplitudes during exposure to 10 μM cGMP. In this and each of the following examples the amplitude histogram was free of points that might indicate the presence of more than one channel. The solid line represents the sum of two gaussian distributions fitted to the data. The channel undergoes transitions from a closed state to an open state with a mean current amplitude of −1.44 pA at this voltage. In the presence of 10 μM cGMP the opening probability (Po) estimated from the areas under the fitted curves was 0.43. The I(V) curve for single channel currents was measured by varying the pipette potential and is linear in the range −50 to +60 mV in symmetrical Na+ solutions with an intercept near zero mV and a slope conductance of 47pS (Fig. 3 A). There was no obvious effect of pipette potential on opening probability.

Bottom Line: There is no apparent effect of voltage on opening probability.In contrast, cAMP failed to activate the channel at concentrations as high as 100 microm.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and the Hopkins Marine Station, Stanford University, Pacific Grove, California 93950, USA. stuartt@leland.stanford.edu

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.

Show MeSH
Related in: MedlinePlus