Developmental expression of the novel voltage-gated sodium channel auxiliary subunit beta3, in rat CNS

Abstract

1. We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Nabeta1.1 and Nabeta2.1 and the novel, recently cloned subunit, beta3. 2. In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and beta3 mRNA at embryonic stages whilst Nabeta1.1 and Nabeta2.1 mRNA was absent throughout this period. 3. Nabeta1.1 and Nabeta2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. beta3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum. 4. Emulsion-dipped slides showed co-localisation of beta3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction. 5. Co-expression in Xenopus oocytes revealed that beta3 could modify the properties of Nav1.3; beta3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation. 6. In conclusion, beta3 is shown to be the predominant beta subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage-gated sodium channels during development.

Figure 1. Sagittal section autoradiographs of whole rat embryo after in situ hybridisation with antisense probes for Nav1.2 (a), Nav1.3 (b), Nav1.6 (c), Naβ1.1 (d), Naβ2.1 (e) and β3 (f) at the stages E10, E15, E17 and E19

Scale bar 7.4 mm. A second probe designed to a different region of the gene for each subunit was used as control. Identical patterns of distribution were observed for both probes for each subunit. The brain at E19 - probe 2 (box) was magnified to show detailed structure whilst the diagram (h) shows the main anatomical regions. Scale bar 2 mm. Sense probe for β3 (g) at E19 shows an example of background levels of hybridisation. Tel, telencephalon; Mesen, mesencephalon; Myelen, myelencephalon; Cor, cortex; Sup coll, superior colliculus; Inf coll, inferior colliculus; Cer flex, cervical flexure; Cereb, cerebellum; Diff hipp, diffentiating hippocampus, Cor plate, cortical plate; Int cor layer, intermediate cortical layer; Cor, cortical neuroepithelium; Olf bulb, olfactory bulb; Acb, nucleus accumbens; Thal nuc, thalamic nuclei; Hypothal nuc, hypothalamic nuclei.

Figure 2. Bright field photomicrographs showing cellular distribution of voltage- gated sodium channel mRNAs in the developing hippocampus at embryonic ages E15

Silver grains demonstrate message for Nav1.2 (a), Nav1.3 (b), Nav1.6 (c), Naβ1.1 (d), Naβ2.1 (e) and β3 (f). Scale bar 10 μm.

Figure 3. Nav1.3 and β3 mRNAs are co-localized in the same cell groups in rat embryo

Bright field high-power photomicrographs of 5 μm thick emulsion-dipped sections, demonstrate the co-expression of Nav1.3 (a) and β3 (b) mRNA in specific neurones (arrowheads) and cell groups of the differentiating field of the thalamus and brain stem at stage E15. Red asterisks indicate orientation markers. Scale bar 10 μm.

Figure 4. In situ hybridisation of voltage-gated sodium channels on sagittal sections of rat brain

X-ray autoradiographs of sections after hybridisation with ³⁵S-labelled antisense probe for Nav1.2 (a), Nav1.3 (b), Nav1.6 (c), Naβ1.1 (d), Naβ2.1 (e) and β3 (f) at postnatal stages P1, P3, P9, P14 and adult. Scale bar 5.5 mm.

Figure 5. Bright field emulsion photomicrographs showing mRNA expression for voltage-gated sodium channel subunits in the hippocampal CA1 neurones at ages P1, P3, P9, P14 and adult

Silver grains demonstrate message for Nav1.2 (a), Nav1.3 (b), Nav1.6 (c), Naβ1.1 (d), Naβ2.1 (e) and β3 (f). Scale bar 20 μm.

Figure 6. Bright field emulsion photomicrographs showing mRNA expression for voltage-gated sodium channel subunits in the hippocampal CA3 neurones at ages P1, P3, P9, P14 and adult

Silver grains demonstrate message for Nav1.2 (a), Nav1.3 (b), Nav1.6 (c), Naβ1.1 (d), Naβ2.1 (e) and β3 (f). Scale bar 20 μm.

Figure 7. Bright field emulsion photomicrographs showing mRNA expression for voltage-gated sodium channel subunits in the hippocampal dentate gyrus neurones at ages P1, P3, P9, P14 and adult

Silver grains demonstrate message for Nav1.2 (a), Nav1.3 (b), Nav1.6 (c), Naβ1.1 (d), Naβ2.1 (e) and β3 (f). Scale bar 20 μm.

Figure 8. Dark field emulsion photomicrographs showing mRNA expression for voltage-gated sodium channel subunits in the hippocampal formation in the adult

Silver grains demonstrate message for Nav1.2 (a), Nav1.3 (b), Nav1.6 (c), Naβ1.1 (d), Naβ2.1 (e) and β3 (f). Scale bar 300 μm.

Figure 9. Co-expression of Nav1.3 with β3 subunit modifies inactivation kinetics

A, Na⁺ currents recorded from Xenopus oocytes expressing Nav1.3 and Nav1.3 +β3. Na⁺ currrents were evoked by applying depolarizing pulses in 5 mV increments between -60 and +40 mV from a holding potential of -100 mV. B, normalized Na⁺ currents from oocytes expressing Nav1.3 and Nav1.3 +β3. Currents evoked by a pulse to -10 mV were normalized to peak amplitudes.

Figure 10

A, effect of β3 on time course of recovery from inactivation of Nav1.3. Peak current amplitudes measured during the test pulse were normalized to the peak currents evoked during the inactivating pulse and plotted as a function of conditioning pulse duration. Data were fitted with a double exponential equation. Nav1.3, ○; Nav1.3 +β3, •. See Table 2 for fit parameters. Inset illustrates typical Nav1.3 currents evoked following conditioning pulses of 20 and 200 ms duration. Following a 20 ms recovery period the Nav1.3 current is dominated by the rapidly inactivating component, while after a 200 ms recovery period there is also a large slowly inactivating component. B, effect of β3 on voltage dependence of activation and inactivation. Conductance-voltage relationships for steady-state activation and inactivation. For steady-state inactivation, peak current amplitudes evoked by the test pulse were normalized to the maximum peak current amplitude and plotted as a function of conditioning pulse potential. Data were fitted with a double Boltzmann function. The steady-state activation data were normalized to peak amplitudes and fitted with a single Boltzmann function. Nav1.3, •; Nav1.3 +β3, ○. See Table 2 for fit parameters. Inset illustrates typical Nav1.3 currents evoked following conditioning pulses to -40 and -70 mV are shown. Following a -70 mV pulse both fast and slow inactivating components are present, while after a -40 mV pulse only the slow inactivating component can be detected.