Contactin associates with Na+ channels and increases their functional expression

Abstract

Contactin (also known as F3, F11) is a surface glycoprotein that has significant homology with the beta2 subunit of voltage-gated Na(+) channels. Contactin and Na(+) channels can be reciprocally coimmunoprecipitated from brain homogenates, indicating association within a complex. Cells cotransfected with Na(+) channel Na(v)1.2alpha and beta1 subunits and contactin have threefold to fourfold higher peak Na(+) currents than cells with Na(v)1.2alpha alone, Na(v)1.2/beta1, Na(v)1.2/contactin, or Na(v)1.2/beta1/beta2. These cells also have a correspondingly higher saxitoxin binding, suggesting an increased Na(+) channel surface membrane density. Coimmunoprecipitation of different subunits from cell lines shows that contactin interacts specifically with the beta1 subunit. In the PNS, immunocytochemical studies show a transient colocalization of contactin and Na(+) channels at new nodes of Ranvier forming during remyelination. In the CNS, there is a particularly high level of colocalization of Na(+) channels and contactin at nodes both during development and in the adult. Contactin may thus significantly influence the functional expression and distribution of Na(+) channels in neurons.

Fig. 1.

Association of contactin and Na⁺ channels in brain and contactin expression in transfected cells. a, Coimmunoprecipitation of the brain lysate with anti-Na⁺ channel antibodies, probed with anti-contactin antibodies. Lane 1, Nonimmune IgG beads as a control; lane 2, anti-Na⁺ channel (NaChα) antibodies; lane 3, anti-Na⁺ channel antibodies preabsorbed with the peptide antigen; lane 4, lysate. The bands at ∼135 kDa represent contactin. Equal amounts of lysate were used in lane 4 and in the coimmunoprecipitation reaction. The dark bands at lower molecular weight in lanes 1–3 represent the antibody used for immunoprecipitation. b, Coimmunoprecipitation of brain with anti-contactin, probed with anti-Na⁺ channel antibodies. Left lane, Nonimmune IgG; right lane, anti-contactin. c–g, Analysis of contactin expression in transfected cells. c, Western blots of cell lines probed with anti-contactin antibodies.Lanes 1 and 2, Two clones of Na_v1.2/β1/contactin; lane 3, Na_v1.2/contactin; lane 4, 1610 cells (untransfected). d–g, Immunocytochemistry of contactin expressed in transfected cells. d, Na_v1.2/contactin. e, f, Two clones of Na_v1.2/β1/contactin. g, Untransfected 1610 cells. Scale bar, 10 μm.

Fig. 2.

Na⁺ currents from transfected cells. Families of currents were recorded under whole-cell patch clamp at different HP: left, −70 mV;right, −90 mV, except for Na_v1.2/β1/β2/contactin, which were at −110 mV. Test voltages were varied in increments of 10 mV within the following ranges (chosen to bracket the maximum peak current, which was generally recorded close to 0 mV): Na_v1.2, Na_v1.2/contactin, Na_v1.2/β1/contactin, −30 to +30 (HP of −70 mV), −50 to +30 (HP of −90 mV); Na_v1.2/β2/contactin, −20 to +20 (HP of −70 mV), −20 to +20 (HP of −90 mV); Na_v1.2/β1, Na_v1.2/β1/β2, −20 to +30 (HP of −70 mV), −40 to +30 (HP of −90 mV); Na_v1.2/β1/β2/contactin, −20 to +30 (HP of −70 mV), −30 to +40 (HP of −110 mV).

Fig. 3.

Na⁺ channel expression in transfected CHL cells. a, Maximum Na⁺current density, plotted as picoamperes per picofaradays of cell capacitance, for the cell lines indicated along theabscissa. The number of cells tested for each transfection paradigm was as follows: Na_v1.2, 20; Na_v1.2/contactin, 19; Na_v1.2/β1, 30; Na_v1.2/β1/β2, 28; Na_v1.2/β1/contactin, 43; Na_v1.2/β2/contactin, 10; Na_v1.2/β1/β2/contactin, 8. The Na_v1.2/β1/contactin result included three different clones of this transfection combination. b, The percentage of ³H-STX binding relative to that for Na_v1.2/contactin cells, to the cell lines indicated along the abscissa. The data for Na_v1.2/β1/contactin, Na_v1.2/β2/contactin, and Na_v1.2/β1/β2/contactin include measurements on three different clones. In both a and b, results for Na_v1.2/β1/contactin were significantly different from those for Na_v1.2/β1 or Na_v1.2/contactin cells (p < 0.005).

Fig. 4.

Association of contactin with Na⁺ channel subunits. Lysates of cells transfected with contactin plus one Na⁺ channel subunit were analyzed by coimmunoprecipitation. In each case, lanescorrespond to beads with nonimmune IgG controls and antibodies, as marked. Western blots were probed with anti-contactin. Left, CHL/1610 cells transfected with Na_v1.2α and contactin, immunoprecipitated (IP) with anti-Na⁺ channel α-subunit antibodies (NaChα). Middle, Cells transfected with β2 subunits and contactin, immunoprecipitated with anti-β2.Right, Cells transfected with β1 subunits and contactin, immunoprecipitated with anti-β1_in antibodies.

Fig. 5.

Immunofluorescence localization of Na⁺ channels (green) and contactin (red) in the PNS. a, b, Adult nodes of Ranvier.a, At ∼95% of sites, contactin was below the level of detection within the nodal gap occupied by Na⁺channels. b, Approximately 5% of adult nodes had a low level of contactin label colocalizing with Na⁺channels. c–e, Demyelinated axons. c,Left, A demyelinated axon at 9 dpi with contactin immunofluorescence (red) widely distributed over its surface. c, Right, Early remyelination: an adherent Schwann cell labeled with anti-MAG (blue) at 9 dpi. Clusters of contactin (red) are visible at the Schwann cell edges. d, A single process cluster of Na⁺channels, with the Schwann cell process at the left. Contactin extends into the region occupied by Na⁺channels; 12 dpi. e, A new node of Ranvier at 12 dpi with contactin primarily paranodal. Scale bars, 10 μm.

Fig. 6.

The frequency of occurrence of three different patterns of expression of contactin at remyelinating nodes. Sites were defined as regions containing a cluster of Na⁺channels. Filled squares, Contactin only at paranodes.Filled circles, Contactin exclusively nodal. Filled triangles, contactin extending from the paranode through the nodal gap.

Fig. 7.

Colocalization of contactin and Na⁺ channels in the CNS. Left column, Immunolabel as indicated in each panel (green);middle column, contactin (red); right column, merge. A, Cryosectioned myelinated axons from large cortical cells in adult rat brain. Numerous nodes of Ranvier are visible and are stained for Na⁺ channels and contactin. Scale bar, 10 μm. B, Adult rat optic nerve, double-labeled for Caspr/contactin (a–c) and Na⁺ channels/contactin (d–f). Scale bars, 5 μm. C, Postnatal day 13 rat optic nerve cryosections double-labeled for Na⁺channels/contactin (a–c) and neurofilaments/contactin (d–f). Scale bars, 10 μm.