Nonmuscle myosin II isoforms interact with sodium channel alpha subunits

Figures

• 

  Figure 1.. Interaction of myh9 with Naα subunits expressed in the mouse brain tissues. WT type (In, lane 1 in (a) and (b)) or Nav1.6 KO (Scn8amedtg; In, lane 1 in (c)) mouse brain tissue lysates were PC with mouse IgG2b isotypes (PC, lane 2 in (a), (b), and (c)) prior to IP using mouse anti-myh9 antibodies (IP, lane 3 in (a), (b), and (c)) of the IgG2b isotypes. Myh9 coimmunoprecipitated pan-Nav ((i) in (a)), Nax ((ii) in (a)), and Nav1.2 ((iv) in (a)) subunits, but not Nav1.1 subunits ((iii) in (a)), expressed in mouse brain tissues. Nav1.6 subunits ((i) in (b)) were not coimmunoprecipitated with myh9 expressed in the mouse brain tissues. As expected, there was not any Nav1.6 immunoreactivity from Scn8amedtg mouse brain tissues ((i) in (c)). Myh9 also coimmunoprecipitated β-actin ((v) in (a) and (ii) in (b) and (c)) and MRLCs ((iii) in (b) and (c)) expressed in mouse brain tissues. The MRLC immunoreactive signal from the mouse brain tissue lysates is barely detectable. Mouse IgG-HC (panel (ii) in (b) and (c)) and IgG-LC (panel (iii) in (b) and (c)), which are separated from their intact immunoglobulins (i.e., used for PC or IP) upon denaturation, could be seen as these blot sections are…

• 

  Figure 2.. Interaction of myh9 and myh10 with Nav subunits expressed in the rat brain tissues. (a) Myh9 and myh10 interact with pan-Nav subunits expressed in the rat brain tissues. Rat brain tissue lysates (In, lanes 1 and 9) were PC with mouse IgG2b isotypes (PC, lanes 2 and 8) and mouse IgG1 isotypes (PC, lane 6) prior to IP using indicated antibodies (lane 3 = myh9, lanes 4 and 10 = myh10, lane 5 = KIF5B, and lane 7 = AnkG) of the same isotypes as those were used for preclearing. IP complexes in the gel were loaded following the loading of their respective isotype (PC) complexes. Pan-Nav subunits (i) were coimmunoprecipitated with myh9, myh10, KIF5B, and AnkG expressed in the rat brain tissues. CoIP of pan-Nav subunits by AnkG and KIF5B served as positive controls. Myh9 and myh10 coimmunoprecipitated β-actin (ii) and MRLCs (iii) expressed in the rat brain tissues. Anti-MRLC antibodies poorly detect their antigens from rat brain tissue lysates (lane 1 in (iii)). AnkG also coimmunoprecipitated β-actin and MRLCs expressed in the rat brain tissues. Denatured mouse IgG-HC (iii) separated from their intact immunoglobulins (i.e., used for PC or IP) could be seen in the immunoblot, as this…

• 

  Figure 3.. Interaction of myh9 and myh10 with Nav1.3 subunits expressed in the postnatal day 3 (P3) rat brain tissues. P3 rat brain tissue lysates (In, lane 1) were PC with mouse IgG2b isotypes (PC, lane 2) prior to IP using mouse anti-myh9 (IP, lane 3) and mouse anti-myh10 (IP, lane 4) antibodies of the IgG2b isotypes. Loading of mIgG2b (PC) complexes in the gel preceded those of the IP complexes. Myh9 and myh10 coimmunoprecipitated Nav1.3 subunits (i) expressed in P3 rat brain tissues. Myh9 and myh10 also coimmunoprecipitated β-actin (ii) and MRLCs (iii) expressed in P3 rat brain tissues. The MRLC immunoreactive signal from the rat tissue lysates is barely detectable and is indicated with an asterisk (*). The denatured mouse IgG-LC (iii) separated from their intact immunoglobulins (i.e., used for PC or IP) could be seen in the immunoblot, as this section of the blot is probed with mouse anti-MRLC antibodies. IgG-LC: immunoglobulin light chain; In: lysate input; IP: immunoprecipitation; mIgG2b: mouse immunoglobulin isotype 2b; MRLC: myosin regulatory light chain; myh: myosin heavy chain; Nav: voltage-sensitive sodium channel; PC: precleared.

• 

  Figure 4.. Interaction of myh9 and myh10 with Nav subunits expressed in adult rat DRG tissues. (a) Interaction of myh9 and myh10 with Nav subunits expressed in adult rat DRG tissues. Adult rat DRG tissue lysates (In, lane 1) were PC with mouse IgG2b isotypes (PC, lane 2) prior to IP using mouse anti-myh9 (IP, lane 3), anti-myh10 (IP, lane 4), and anti-KIF5B antibodies of IgG2b isotypes. Loading of IgG2b (PC) complexes in the gel preceded those of the IP complexes. Myh9 and myh10 coimmunoprecipitated Nav1.7 (i) and Nav1.8 (ii) subunits, but not Nav1.9 (iii) subunits, expressed in rat DRG tissues. As expected, both myh9 and myh10, but not KIF5B, coimmunoprecipitated β-actin expressed in rat DRG tissues. KIF5B also coimmunoprecipitated Nav1.8 subunits, but not Nav1.7 and Nav1.9 subunits, expressed in rat DRG tissues. Mouse IgG-HC (iv) and IgG-LC (iv), which are separated from their intact immunoglobulins upon denaturation, are seen, as this blot section was probed with mouse (anti-β-actin) antibodies. (b) Interaction of Nav1.7 and Nav1.8 subunits with myh9 and myh10 expressed in adult rat DRG tissues. Adult rat DRG tissue lysates (In, lanes 2 and 4) were PC with mouse IgG1 isotypes (PC, lane…

• 

  Figure 5.. Coexpression of myh10 increases peak current density of Nav1.8 channels heterologously expressed in ND7/23 cells. Representative sodium currents were recorded from ND7/23 cells transiently expressing Nav1.8 in the presence of GFP (a) or GFP-myh10 (b). Cells were held at −80 mV, and sodium currents were elicited by a series of step depolarizations from −60 to +50 mV in 5-mV increments. (c) Coexpression of myh10 hyperpolarized activation of Nav1.8. (d) Coexpression of myh10 enhanced steady-state fast inactivation of Nav1.8. (e) Coexpression of myh10 reduced the amplitude of the ramp currents. GFP: green fluorescent protein; myh: myosin heavy chain.

• 

  Figure 6.. A model for interaction for NM-IIs with mammalian sodium channel α subunits. NM-IIs could be interacting with Naα subunits directly (i) or via adaptor proteins (such as ankyrins) (ii). Also shown in the model the canonical spectrin–ankyrin–Nav signaling network. NM-II: nonmuscle myosin II.