Glycosylation alters steady-state inactivation of sodium channel Nav1.9/NaN in dorsal root ganglion neurons and is developmentally regulated - PubMed (original) (raw)

Glycosylation alters steady-state inactivation of sodium channel Nav1.9/NaN in dorsal root ganglion neurons and is developmentally regulated

L Tyrrell et al. J Neurosci. 2001.

Abstract

Na channel NaN (Na(v)1.9) produces a persistent TTX-resistant (TTX-R) current in small-diameter neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Na(v)1.9-specific antibodies react in immunoblot assays with a 210 kDa protein from the membrane fractions of adult DRG and trigeminal ganglia. The size of the immunoreactive protein is in close agreement with the predicted Na(v)1.9 theoretical molecular weight of 201 kDa, suggesting limited glycosylation of this channel in adult tissues. Neonatal rat DRG membrane fractions, however, contain an additional higher molecular weight immunoreactive protein. Reverse transcription-PCR analysis did not show additional longer transcripts that could encode the larger protein. Enzymatic deglycosylation of the membrane preparations converted both immunoreactive proteins into a single faster migrating band, consistent with two states of glycosylation of Na(v)1.9. The developmental change in the glycosylation state of Na(v)1.9 is paralleled by a developmental change in the gating of the persistent TTX-R Na(+) current attributable to Na(v)1.9 in native DRG neurons. Whole-cell patch-clamp analysis demonstrates that the midpoint of steady-state inactivation is shifted 7 mV in a hyperpolarized direction in neonatal (postnatal days 0-3) compared with adult DRG neurons, although there is no significant difference in activation. Pretreatment of neonatal DRG neurons with neuraminidase causes an 8 mV depolarizing shift in the midpoint of steady-state inactivation of Na(v)1.9, making it indistinguishable from that of adult DRG neurons. Our data show that extensive glycosylation of rat Na(v)1.9 is developmentally regulated and changes a critical property of this channel in native neurons.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Immunoblot assay to show specificity of anti-Nav1.9 antibody. A, An immunoreactive band of molecular weight ∼210 kDa is detected in DRG and trigeminal (Trig.) ganglia but not in spinal cord (Sp. C) or liver. A much smaller nonspecific protein of molecular weight ∼100 kDa is detected in some samples. B, The blot was stripped and reprobed with a generic sodium channel antibody. DRG, trigeminal, and spinal cord samples show multiple immunoreactive bands, consistent with the presence of multiple sodium channels in these tissues. A number of smaller proteins are also detected and are the result of nonspecific interaction with the secondary antibody. C, Membrane fractions from adult DRG (Tissue) and from 24 hr cultured DRG cells (Culture). A single immunoreactive band of ∼210 kDa is detected in the cultured DRG sample, consistent with the conclusion that the ∼100 kDa protein is unrelated to Nav1.9. Positions of molecular weight markers 209, 130, and 78 kDa are indicated.

Fig. 2.

Fig. 2.

Axotomy reduces the level of Nav1.9 protein in rat DRG. The sciatic nerves of the right side of four rats were transected, and the level of Nav1.9 protein in the axotomized DRG (A) was compared with that in the intact ganglia (C) 14 d after axotomy in an immunoblot assay. The L4 and L5 DRG from the intact (C) and from the axotomized (A) sides were pooled from two rats, and the membrane fraction was analyzed using the Nav1.9-specific antibody. The intensity of the bands was determined by densitometry. Axotomy results in a clear reduction of the level of Nav1.9 protein in axotomized DRG. The nonspecific ∼100 kDa protein serves as a convenient marker to demonstrate equal loading of the gel. Positions of molecular weight markers 217 and 123 kDa are indicated.

Fig. 3.

Fig. 3.

Two Nav1.9 immunoreactive bands are present in neonatal rat DRG. A, Membrane fractions from DRG and trigeminal ganglia (Trig) of P3 and adult (Ad) rats were probed with the Nav1.9 antibody and show an additional, higher molecular weight immunoreactive protein in the P3 sample compared with the adult sample. B, DRG and trigeminal ganglia membrane fractions from P0 and P7 rats contain two immunoreactive proteins. Trace amount of the slower migrating band is present in the E17 sample, whereas the P21 DRG sample contains only the faster migrating species.

Fig. 4.

Fig. 4.

Both Nav1.9 immunoreactive proteins are glycosylated. A, Treatment of the membrane fractions from P0, P2, and P4 DRG with the glycosidase PNGase F (Enz) converts the immunoreactive proteins into a single faster migrating species. Samples were separated on a 4–15% gradient gel before electroblotting. Ad shows the immunoreactive protein from untreated adult DRG sample. B, Similar treatment of the membrane fraction from adult DRG converts the ∼210 kDa protein into a faster migrating species. Samples were separated on a 5% gel before electroblotting. The + and − signs indicate treated and untreated samples, respectively.

Fig. 5.

Fig. 5.

Separation of Nav1.9 Na+ currents from Nav1.8 Na+ currents by prepulse subtraction in adult and neonatal DRG neurons. A, Families of Na+ current traces from an adult small-diameter DRG neuron in the presence of 300 n

m

TTX. The capacitance of the adult DRG neuron was 13.31 pF. Both Nav1.9 Na+ current and Nav1.8 Na+ current were elicited in response to 100 msec test pulses from a holding potential of −130 mV to test potentials ranging from −100 to + 60 mV in 5 mV steps. B, The same neuron when given a prepulse of −50 mV for 500 msec before the test potentials from −100 to +60 mV elicited only Nav1.8 Na+ currents. C, Subtraction of current traces shown in B from the current traces shown in A yields Nav1.9 Na+currents. D–F represent separation of Nav1.9 Na+ currents from Nav1.8 Na+ currents in a neonatal DRG neuron as shown in A–C. The capacitance of the neonatal DRG neuron was 7.13 pF.

Fig. 6.

Fig. 6.

Desialidation affects the steady-state inactivation of Nav1.9 Na+ currents only in neonatal DRG neurons. A, Normalized current–voltage curves for neonatal (filled circles), adult (open circles), neonatal desialidated (filled squares), and adult desialidated (open squares) Nav1.9 Na+currents are shown in A. Current values were normalized to the peak value obtained at −40 mV. Symbols are the same for B and C and are shown also in the figure for clarity. B, Desialidation did not affect voltage dependence of activation of neonatal Nav1.9 Na+ currents. Voltage dependence of activation of neonatal, adult, neonatal desialidated, and adult desialidated Nav1.9 channels are shown. C, Desialidation causes a depolarizing shift of steady-state inactivation of neonatal Nav1.9 Na+ currents. Steady-state voltage dependence of inactivation of neonatal, adult, neo- natal desialidated, and adult desialidated Nav1.9 channels are shown. Lines are the Boltzmann fits to the data. The values of_V_1/2 and slope from Boltzmann fits of steady-state activation and steady-state inactivation of pooled neonatal, adult, neonatal desialidated, and adult desialidated Nav1.9 Na+ currents are shown in Table1.

Similar articles

Cited by

References

    1. Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature. 1996;379:257–262. - PubMed
    1. Barchi RL, Cohen SA, Murphy LE. Purification from rat sarcolemma of the saxitoxin-binding component of the excitable membrane sodium channel. Proc Natl Acad Sci USA. 1980;77:1306–1310. - PMC - PubMed
    1. Beckh S, Noda M, Lübbert H, Numa S. Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development. EMBO J. 1989;8:3611–3616. - PMC - PubMed
    1. Belcher SM, Zerillo CA, Levenson R, Ritchie JM, Howe JR. Cloning of a sodium channel alpha subunit from rabbit Schwann cells. Proc Natl Acad Sci USA. 1995;92:11034–11038. - PMC - PubMed
    1. Bennett E, Urcan MS, Tinkle SS, Koszowski AG, Levinson SR. Contribution of sialic acid to the voltage dependence of sodium channel gating. A possible electrostatic mechanism. J Gen Physiol. 1997;109:327–343. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources