ADAM22, a Kv1 channel-interacting protein, recruits membrane-associated guanylate kinases to juxtaparanodes of myelinated axons

Abstract

Clustered Kv1 K(+) channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments, and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 alpha subunits, and then used mass spectrometry to identify interacting proteins. We found that a disintegrin and metalloproteinase 22 (ADAM22) is a component of the Kv1 channel complex and that ADAM22 coimmunoprecipitates Kv1.2 and the membrane-associated guanylate kinases (MAGUKs) PSD-93 and PSD-95. When coexpressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, coexpression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel-interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that, like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K(+) channel complex that recruits MAGUKs to juxtaparanodes.

Figure 1.

ADAM22 is part of the Kv1 channel protein complex. A, Silver-stained gel of a Kv1.2 immunoprecipitate. The asterisk indicates the Kv1.2 band, and the arrowhead indicates the ADAM22 band. B, Tandem mass spectra obtained from precursor ions with m/z = 629.8495⁺² (top) and 719.3796⁺² (bottom), corresponding to two peptides spanning the residues Leu-383 to Lys-393, and Ser-466 to Arg-479, respectively, of rat ADAM22. The observed sequence ions are labeled. Ions corresponding to neutral losses are marked with stars.

Figure 2.

ADAM22 interacts with Kv1 channels, MAGUKs, and Lgi proteins. A, Immunoblot analysis of coimmunoprecipitation reactions using antibodies against Kv1.2, PSD-95, PSD-93, ADAM22, and pan-Neurofascin (PanNF). The input lane corresponds to the detergent soluble fraction, whereas the remainder of the protein is shown in the insoluble fraction (insol). B, Cotransfection of ADAM22 and Kv1.4 in COS7 cells shows no surface clustering. C, D, Cotransfection of ADAM22, Kv1.4, and PSD-95 (C) or PSD-93 (D) results in the formation of large surface clusters that can be detected using antibodies directed against an extracellular epitope of ADAM22. Scale bar, 20 μm.

Figure 3.

ADAM22 colocalizes with Kv1.2 and MAGUKs at juxtaparanodes, BCTs, and the AIS. A–D, Immunostaining for ADAM22 (A, D), PSD-93 (B), and PSD-95 (C) demonstrates colocalization with Kv1.2 (red) at juxtaparanodes in the CNS (A–C) and PNS (D). Caspr immunoreactivity (blue) defines the paranodal junctions. E, F, ADAM22 immunoreactivity colocalizes with Kv1.2 (E) and PSD-95 (F) at BCTs. PSD-93 (F) is enriched in somatodendritic domains of Purkinje neurons. In E, neurotracer (NT) fluorescence indicates the location of neuronal cell bodies and shows that the BCT lies below the Purkinje neuron (inset). G, In cultured hippocampal neurons, ADAM22 immunostaining (green) colocalizes with βIV spectrin (red) at the AIS and is excluded from somatodendritic domains indicated by MAP2 immunoreactivity (blue). H, ADAM22 immunoreactivity (green) colocalizes with Kv1.1 (red) and βIV spectrin (blue) at the AIS. Scale bars: A–D, 10 μm; E, F, 100 μm; E, F, inset; G, H, 20 μm.

Figure 4.

Coexpression of ADAM22 and PSD-95 with Kv1.2 does not affect the voltage-dependent activation and deactivation kinetics. A, Current traces from COS cells transfected with Kv1.2 alone (0.1 μg) (top) or in combination with ADAM22 and PSD-95 (0.5 μg each) (bottom). Voltage steps were applied from holding potential −100 to +70 mV in 10 mV increments. B, I–V curve with Kv1.2 alone (filled squares) or with in combination of ADAM22 and PSD-95 (open circles). C, Voltage-dependent activation. The lines represent the single Boltzmann fits to the mean ± SE normalized data. D, Activation constant as a function of voltage. Current traces were fitted with a single exponential function from the time they reached half level to the maximum current. E, Deactivation constant. Cells were held at −100 mV and depolarized to 30 mV for 80 ms followed by test pulses to variable potentials from −100 to 0 mV.

Figure 5.

MAGUKs are not required for clustering of ADAM22 at juxtaparanodes. A–G, Immunostaining of myelinated CNS axons for ADAM22 (green), Kv1.2 (red), and βIV spectrin or Caspr (blue) in WT, Caspr^−/−, Caspr2^−/−, PDS-93^−/−, PSD-95^−/−, and PSD-95/PSD-93^−/− mice.

Figure 6.

PSD-95 is required for clustering of ADAM22 at BCTs. A, Immunostaining of BCTs for ADAM22 (green) and Kv1.2 (red) in PSD-95^−/−, PSD-93^−/−, PSD-95/PSD-93 double −/−, and Caspr2^−/− mice. B, High-magnification images showing immunostaining of BCTs for ADAM22 (green) and Kv1.2 (red), and Purkinje neuron AIS (βIV spectrin; blue) in PSD-95^−/−, PSD-93^−/−, PSD-93/PSD-95^−/−, and Caspr2^−/− mice. Scale bar, 10 μm.

Figure 7.

ADAM22 is required for clustering of PSD-93 and PSD-95 at juxtaparanodes. Immunostaining of P10 WT and ADAM22-null (ADAM22^−/−) myelinated spinal cord and brainstem axons. In the spinal cord, nodes are identified by βIV spectrin immunoreactivity (blue). A, ADAM22 (red), Kv1.2 (green), and βIV spectrin (blue). B, ADAM22 (red), Caspr2 (green), and βIV spectrin (blue). C, PSD-93 (red), Kv1.2 (green), and βIV spectrin (blue). D, PSD-95 (red), Kv1.2 (green), and βIV spectrin (blue). Scale bar, 5 μm.

Figure 8.

ADAM22 is not required for clustering of Kv1 channels at axon initial segments. Immunostaining of axon initial segments using antibodies against Kv1.2 (green) and AnkG (red) in WT, Caspr^−/−, Caspr2^−/−, ADAM22^−/−, PSD-93^−/−, PSD-95^−/−, and PSD-93/PSD-95^−/− mice. Scale bar, 10 μm.