I SA channel complexes include four subunits each of DPP6 and Kv4.2

Abstract

Kv4 potassium channels produce rapidly inactivating currents that regulate excitability of muscles and nerves. To reconstitute the neuronal A-type current I(SA), Kv4 subunits assemble with DPP6, a single transmembrane domain accessory subunit. DPP6 alters function-accelerating activation, inactivation, and recovery from inactivation-and increases surface expression. We sought here to determine the stoichiometry of Kv4 and DPP6 in complexes using functional and biochemical methods. First, wild type channels formed from subunit monomers were compared with channels carrying subunits linked in tandem to enforce 4:4 and 4:2 assemblies (Kv4.2-DPP6 and Kv4.2-Kv4.2-DPP6). Next, channels were overexpressed and purified so that the molar ratio of subunits in complexes could be assessed by direct amino acid analysis. Both biophysical and biochemical methods indicate that I(SA) channels carry four subunits each of Kv4.2 and DPP6.

FIGURE 1.

DPP6 impacts current magnitude in channels with fixed subunit ratios. A, schematic of subunits and representative current traces at +40 mV for channels assembled with K and D or the linked subunits KKD and KD measured in Xenopus oocytes by two-electrode voltage clamp. B, relative peak current density at +40 mV for various pore-forming subunits in the absence and presence of D. These were calculated from three to six different groups of oocytes, each group with 5–16 cells. The raw currents from one group (μA mean ± S.E.) at +40 mV with indicated subunits were: K, 0.64 ± 0.15 (n = 10); K + D, 8.28 ± 0.5 (n = 6); KK, 0.64 ± 0.09 (n = 8); KK + D, 6.26 ± 0.46 (n = 9); KKD, 0.78 ± 0.05 (n = 7); KKD + D, 8.23 ± 0.81 (n = 8); KD, 3.4 ± 0.2 (n = 9); KD + D, 10.2 ± 0.4 (n = 7).

FIGURE 2.

Natural and KD channels show similar kinetic properties. Normalized and superimposed representative current traces at 0 mV for channels with D (filled symbols, dark lines) or without D (open symbols, gray lines) and the pore-forming subunits K, KK, KD, and KKD. A, K versus K + D. B, KK versus KK + D. C, KKD versus KKD + D. D, KD versus KD + D. E, overlay of KD and K + D; inactivation fast component for traces shown: K + D and KD, τ_fast = 13.4 and 11.8 ms, respectively.

FIGURE 3.

Biophysical properties of natural and engineered channels. A, conductance-voltage relationships, steady state inactivation-voltage relationships, and time to peak for K (open square), KKD (open triangle), KD (open diamond), and K + D(filled square) for biophysical parameters listed in Table 1. The insets show voltage protocol as described under “Experimental Procedures” with arrowheads to indicate where current was measured. B, assembly with monomer D (filled symbols) shifts KKD channel (triangle) G-V and steady state inactivation-voltage relationships and time to peak (dashed line from data in A) to those measured for natural K + D(square) and KD (diamond) channels. The biophysical parameters are listed in Table 1.

FIGURE 4.

Amino acid analysis of naturally assembled channels. Purification of channels formed in COS7 cells with K, D, and KChIP2 subunits was achieved via a 1d4 epitope on K after treatment with DTBP. Purified proteins were subjected to reducing conditions to reverse DTBP linkages and separated by SDS-PAGE. A, proteins detected by Western blotting with anti-1d4 (Kv4.2–1d4), anti-HA (DPP6-HA), and anti-KChIP2 monoclonal antibodies. B, proteins detected with Coomassie Brilliant Blue. Bands corresponding to K and D were excised and subjected to amino acid analysis (Table 2). Bands a, b, c, and d co-purified and were identified by mass spectrometry to be a 90-kDa heat shock protein, a tubulin, a trypsin precursor, and an actin, respectively (data not shown).