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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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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 (NPo) as a function of time before  and after adding 1 mM 8-br-cGMP. NPo 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.
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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 (NPo) as a function of time before and after adding 1 mM 8-br-cGMP. NPo 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.

Mentions: Single channel currents were recorded in cell attached patch configuration during the application of 8-br-cGMP, a membrane permeable analogue of cGMP. The pipette solution contained 153 mM NaCl, 10 mM EGTA, 1 mM EDTA, 1 μM TTX, and 10 HEPES (pH 7.4; 30°C), and the potential across the patch was equal to the resting potential, estimated from microelectrode measurements to be between −20 and −40 mV. Fig. 1 A shows records taken before and at successive times after adding 1 mM 8-br-cGMP in the external medium. The cGMP analogue activates a channel permeable to Na+. Channel activity occurs in bursts of brief openings, and the rate of occurrence of bursts as well as the burst length increase with time during exposure to 8-br-cGMP. Amplitude histograms constructed from 10-s segments recorded at various times after adding 8-br-cGMP are shown in Panel B. The peak at zero current represents the background noise. Additional peaks at approximately −0.6 and −1.2 pA begin to appear and increase in amplitude with time. The progressive change in the amplitude histogram indicates that this patch contained at least two inward current channels that are activated by 8-br-cGMP and that each channel contributes a current of ∼0.6 pA. Opening probability for the multiple channels in this patch (NPo) was estimated from the ratio of mean open time to mean closed time using a 50% criterion for detection of unitary events. NPo increased gradually, reaching approximately 0.7 after 14 min (Panel C). The accuracy of this plot is limited because the number of channels in the patch is not known precisely and because brief openings may have been missed due to low pass filtering. Nevertheless, the increase in opening probability follows approximately the same time course as the increase in whole cell inward current measured with perforated patch voltage clamp under similar conditions (Mathes and Thompson, 1996). In both cases, gradual activation probably results from slow entry and gradual intracellular accumulation of the cGMP analogue.


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

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

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 (NPo) as a function of time before  and after adding 1 mM 8-br-cGMP. NPo 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233783&req=5

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 (NPo) as a function of time before and after adding 1 mM 8-br-cGMP. NPo 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.
Mentions: Single channel currents were recorded in cell attached patch configuration during the application of 8-br-cGMP, a membrane permeable analogue of cGMP. The pipette solution contained 153 mM NaCl, 10 mM EGTA, 1 mM EDTA, 1 μM TTX, and 10 HEPES (pH 7.4; 30°C), and the potential across the patch was equal to the resting potential, estimated from microelectrode measurements to be between −20 and −40 mV. Fig. 1 A shows records taken before and at successive times after adding 1 mM 8-br-cGMP in the external medium. The cGMP analogue activates a channel permeable to Na+. Channel activity occurs in bursts of brief openings, and the rate of occurrence of bursts as well as the burst length increase with time during exposure to 8-br-cGMP. Amplitude histograms constructed from 10-s segments recorded at various times after adding 8-br-cGMP are shown in Panel B. The peak at zero current represents the background noise. Additional peaks at approximately −0.6 and −1.2 pA begin to appear and increase in amplitude with time. The progressive change in the amplitude histogram indicates that this patch contained at least two inward current channels that are activated by 8-br-cGMP and that each channel contributes a current of ∼0.6 pA. Opening probability for the multiple channels in this patch (NPo) was estimated from the ratio of mean open time to mean closed time using a 50% criterion for detection of unitary events. NPo increased gradually, reaching approximately 0.7 after 14 min (Panel C). The accuracy of this plot is limited because the number of channels in the patch is not known precisely and because brief openings may have been missed due to low pass filtering. Nevertheless, the increase in opening probability follows approximately the same time course as the increase in whole cell inward current measured with perforated patch voltage clamp under similar conditions (Mathes and Thompson, 1996). In both cases, gradual activation probably results from slow entry and gradual intracellular accumulation of the cGMP analogue.

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