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Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

Patel RR, Barbosa C, Xiao Y, Cummins TR - PLoS ONE (2015)

Bottom Line: We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents.We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1.Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms.

View Article: PubMed Central - PubMed

Affiliation: Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

No MeSH data available.


Related in: MedlinePlus

Rate and fraction of recovery from fast inactivation by hNav1.1 and hNav1.6.A, Representative traces of recovery from fast inactivation measured by first inducing fast inactivation from a holding potential of -100mV with a 20ms step pulse to 0mV and then applying a 20ms test pulse to 0mV subsequent to various recovery times at -70mV. Inset, Protocol used to measure recovery from fast inactivation. B, hNav1.6 (blue circles; n = 17) has a smaller time constant for recovery at -70mV compared to hNav1.1 (black squares; n = 19). C, Maximal fraction recovered from fast inactivation was greater for hNav1.1 at voltage ranging from -90mV to -70mV compared to hNav1.6 (Unpaired t-test, *p < 0.05).
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pone.0133485.g003: Rate and fraction of recovery from fast inactivation by hNav1.1 and hNav1.6.A, Representative traces of recovery from fast inactivation measured by first inducing fast inactivation from a holding potential of -100mV with a 20ms step pulse to 0mV and then applying a 20ms test pulse to 0mV subsequent to various recovery times at -70mV. Inset, Protocol used to measure recovery from fast inactivation. B, hNav1.6 (blue circles; n = 17) has a smaller time constant for recovery at -70mV compared to hNav1.1 (black squares; n = 19). C, Maximal fraction recovered from fast inactivation was greater for hNav1.1 at voltage ranging from -90mV to -70mV compared to hNav1.6 (Unpaired t-test, *p < 0.05).

Mentions: We also compared recovery from fast inactivation (repriming), a property that can limit the channels ability to sustain high firing frequencies. To do this, we held cells at -100mV and applied a prepulse to 0mV for 20ms to induce fast inactivation and then allowed the channels to recover for increasing durations at potentials ranging from -100mV to -70mV before applying a test pulse to 0mV for 20ms to measure the available channels (Fig 3A, inset). Representative traces of recovery from fast inactivation at -70mV are depicted in Fig 3A. Each test pulse current was normalized to the maximal current at each time point and plotted as a function of time for each recovery voltage (S2 Fig). Time constants for repriming kinetics were estimated using single exponential fits and these time constants are plotted as a function of recovery voltage in Fig 3B. We found that hNav1.1 and hNav1.6 had similar time constant values for recovery from inactivation at voltages ranging from -100mV to -80mV. However, at -70mV hNav1.6 (τ = 22.3 ± 1.1ms; n = 17) had significantly faster repriming kinetics compared to hNav1.1 (τ = 33.1 ± 2.1ms; n = 19). It is important to note that the maximal fraction recovered was greater for hNav1.1 compared to hNav1.6 at recovery voltages from -90mV to -70mV (Fig 3C), consistent with the differences observed in the voltage-dependence of steady-state inactivation.


Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

Patel RR, Barbosa C, Xiao Y, Cummins TR - PLoS ONE (2015)

Rate and fraction of recovery from fast inactivation by hNav1.1 and hNav1.6.A, Representative traces of recovery from fast inactivation measured by first inducing fast inactivation from a holding potential of -100mV with a 20ms step pulse to 0mV and then applying a 20ms test pulse to 0mV subsequent to various recovery times at -70mV. Inset, Protocol used to measure recovery from fast inactivation. B, hNav1.6 (blue circles; n = 17) has a smaller time constant for recovery at -70mV compared to hNav1.1 (black squares; n = 19). C, Maximal fraction recovered from fast inactivation was greater for hNav1.1 at voltage ranging from -90mV to -70mV compared to hNav1.6 (Unpaired t-test, *p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4504674&req=5

pone.0133485.g003: Rate and fraction of recovery from fast inactivation by hNav1.1 and hNav1.6.A, Representative traces of recovery from fast inactivation measured by first inducing fast inactivation from a holding potential of -100mV with a 20ms step pulse to 0mV and then applying a 20ms test pulse to 0mV subsequent to various recovery times at -70mV. Inset, Protocol used to measure recovery from fast inactivation. B, hNav1.6 (blue circles; n = 17) has a smaller time constant for recovery at -70mV compared to hNav1.1 (black squares; n = 19). C, Maximal fraction recovered from fast inactivation was greater for hNav1.1 at voltage ranging from -90mV to -70mV compared to hNav1.6 (Unpaired t-test, *p < 0.05).
Mentions: We also compared recovery from fast inactivation (repriming), a property that can limit the channels ability to sustain high firing frequencies. To do this, we held cells at -100mV and applied a prepulse to 0mV for 20ms to induce fast inactivation and then allowed the channels to recover for increasing durations at potentials ranging from -100mV to -70mV before applying a test pulse to 0mV for 20ms to measure the available channels (Fig 3A, inset). Representative traces of recovery from fast inactivation at -70mV are depicted in Fig 3A. Each test pulse current was normalized to the maximal current at each time point and plotted as a function of time for each recovery voltage (S2 Fig). Time constants for repriming kinetics were estimated using single exponential fits and these time constants are plotted as a function of recovery voltage in Fig 3B. We found that hNav1.1 and hNav1.6 had similar time constant values for recovery from inactivation at voltages ranging from -100mV to -80mV. However, at -70mV hNav1.6 (τ = 22.3 ± 1.1ms; n = 17) had significantly faster repriming kinetics compared to hNav1.1 (τ = 33.1 ± 2.1ms; n = 19). It is important to note that the maximal fraction recovered was greater for hNav1.1 compared to hNav1.6 at recovery voltages from -90mV to -70mV (Fig 3C), consistent with the differences observed in the voltage-dependence of steady-state inactivation.

Bottom Line: We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents.We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1.Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms.

View Article: PubMed Central - PubMed

Affiliation: Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

No MeSH data available.


Related in: MedlinePlus