Localisation of NKA

Using vanadate facilitated [3_{H]ouabain, which only detects functional NKA heterodimers,}

the presence of NKA is extensive in both the sarcolemma and the t-tubules of skeletal muscle in various species (Clausen, 2003). While a high concentration of NKA has been confirmed in the sarcolemma (Clausen et al., 1974; Venosa et al., 1981; Clausen, 2003), the methodological problems in isolating the t-tubules for vanadate facilitated ouabain binding have led to some uncertainty quantifying the content of NKA in t-tubules of skeletal muscle. Initial research suggested the existence of NKA in t-tubules using isolated frog single fibres, where Na+ _{and K}+ _{gradients were maintained despite the removal of the sarcolemma}

(Costantin et al., 1967). Lau et al. (1979) used isolated t-tubular membranes from rabbit and confirmed an ATP-driven transport of Na+ _{into the t-tubules, congruent with the concept of} the presence of NKA in the t-tubules. Venosa et al. (1981) attempted to quantify the amount of NKA in t-tubules using vanadate facilitated ouabain binding and only found a 20% decrease in ouabain binding when the t-tubule NKA were inhibited, suggesting that 20% of NKA were in the t-tubules. However, the methodology used by Venosa and Horowicz (1981) involving glycerol pre-treated muscle has been argued to have underestimated the quantity of NKA in the t-tubules (Clausen, 2003). Several studies support the concept of similar overall content of NKA in the t-tubules compared to the sarcolemma in various animal muscles (Barchi et al., 1977; Mitchell et al., 1983; Hidalgo et al., 1986). Further, western blotting of the α2 isoform, the most abundant isoform in skeletal muscle NKA (Hansen, 2001) has confirmed that approximately 50% of the α2 isoforms are located in the t-tubules when measured using membrane fractionation (Kristensen et al., 2010). When the considerably larger area of the t-tubules is taken into account however, the sarcolemma does have a much higher total content of NKA in comparison to the t-tubules (Clausen, 2003).

2.3.5.1 Localisation of NKA isoforms

The NKA α1 isoform is expressed almost entirely in the sarcolemma of both soleus and EDL

in rats (Kristensen et al., 2010) and also in human soleus muscle as confirmed by histological measures (Hundal et al., 1994). In addition, α1 has consistently been reported to

be 2-4 fold higher in rat oxidative muscle compared to glycolytic muscle (Thompson et al., 1996; Juel et al., 2001; Fowles et al., 2004; Kristensen et al., 2010). This result was not

confirmed in humans, with no difference in α1 abundance found between human Type I and

Type II single fibres (Thomassen et al., 2013).

The NKA α2 isoform constitutes ~75-85% of all α isoforms of the NKA in rat EDL muscle

(Hansen, 2001). Several studies using rats have found greater α2 abundance in the EDL

muscle compared to the soleus by as much as 50% (Clausen et al., 1982; Bundgaard et al., 2002; Musch et al., 2002). In human single fibres, Type I fibres had 37% less α2 abundance

(Thomassen et al., 2013). Conversely, other studies found either no difference, or contradictory findings in regard to α2 abundance between fast and slow twitch muscle

fibres in rats (Chin et al., 1993; Fowles et al., 2004). Fowles et al. (2004) found that the EDL muscle had ~40% greater [3_{H]ouabain binding site content compared to the soleus, yet}

puzzlingly reported no significant difference using western blotting, despite that the [3_{H]ouabain binding assay only detects the α}

2 isoform in rat muscle. There was a significant

decrease in α2 abundance using both methodologies in the white gastrocnemius compared

to the red portion (Fowles et al., 2004), although the rats were older than those used by many of the studies finding higher α2 abundance in the EDL (Clausen et al., 1982;

Bundgaard et al., 2002; Musch et al., 2002). With only limited human research, conflicting findings in rats, different methodologies of quantifying NKA and varying ages of rodents used; it is not at this time definitively clear whether the α2 isoform is preferentially

expressed between skeletal muscle fibre type. It is likely however that Type II muscle fibres have a higher relative α2 abundance, as this has been reported in human single muscle

fibres (Thomassen et al., 2013) and has been consistently supported by findings in rat muscle (Clausen et al., 1982; Bundgaard et al., 2002; Musch et al., 2002). Within skeletal

muscle, approximately half of the α2 isoforms were found in the sarcolemma and calveolae

membranes, while the remaining α2 isoforms were found throughout the t-tubule system

(Hundal et al., 1994; Lavoie et al., 1995; Williams et al., 2001; Kristensen et al., 2010)..

The β1 NKA isoform is found in a higher abundance in oxidative compared to glycolytic

muscle fibres, as well as in soleus compared to the EDL in rats (Fowles et al., 2004; Zhang et al., 2006), while the β2 isoform has a higher relative abundance in the EDL and glycolytic

fibres compared to the soleus and oxidative fibres, respectively. The localisation of phospholemman, which regulates the activity of the NKA heterodimer, is distributed throughout both the muscle sarcolemma and the t-tubules and seems to be distributed similarly between muscle types in rats (Rasmussen et al., 2008) and humans (Thomassen et al., 2013). Although one study in rat muscle reported higher content of phospholemman in the sarcolemma of oxidative fibres compared to glycolytic fibres (Juel, 2009). Within skeletal muscle fibres, both β1 and β2 were found to be expressed in both the muscle

sarcolemma and throughout the t-tubules (Hundal et al., 1992; Lavoie et al., 1996). There are approximately 6 times more β isoforms in the cell membrane compared to in the t- tubules (Lavoie et al., 1997). In addition, there was approximately 5-fold more β isoforms in the cell membrane than α isoforms and 2.5 fold more β than α isoforms in the t-tubules (Lavoie et al., 1997). Considering a functional NKA heterodimer requires both α and β subunit, this finding suggests there is a substantial surplus of β subunits in both the membrane and t-tubules of skeletal muscle (Lavoie et al., 1997). The physiological function of these excess β subunit isoforms is not known (Kristensen et al., 2010). The β1 isoform is

than β2 (Lavoie et al., 1997). It should be noted though that it could not be determined what

fraction of β1 or β2 isoforms are actually functional and thus it is unknown how accurate this

ratio of β1: β2 is when functional subunits are considered.

While well described in rats (Lavoie et al., 1997; Fowles et al., 2004; Zhang et al., 2006), the fibre type specificity of NKA content is less clear in humans. Only a single study has investigated NKA isoform abundance in human single muscle fibres and reported 37% lower α2 abundance in Type I fibres than Type II fibres, with no fibre-type difference for the α1 and

β1 isoforms (Thomassen et al., 2013). This is consistent with most rodent studies using

vanadate-facilitated ouabain binding, with 20-50% higher content NKA in rodent EDL in comparison to the soleus (Clausen et al., 1982; Kjeldsen et al., 1984; Bundgaard et al., 2002; Musch et al., 2002). However, It should be emphasized that no assay is available to quantify total functional pumps in single human fibres, as in contrast to the protein abundance of single fibres relative to another as reported by Thomassen et al. (2013). Thus, further research is required to definitively establish the fibre type specificity of NKA isoforms in human skeletal muscle

In conclusion, the key findings in regards to localisation of Na+_,K+_{-ATPase are:}

1) Within the muscle cell, the NKA α1 isoform is expressed only in the sarcolemma, while

the α2 isoform is expressed approximately equally between the sarcolemma and t-tubular

membranes.

3) Most evidence suggests that Type II fibres have a higher abundance of NKA, although this is not definitive and the α1 isoform is 2-4 more abundant in slow twitch compared to fast

twitch muscles.

4) While the α1 and β1 isoforms have an increased affinity for Na+ compared to the α2 and β2

isoforms in basal conditions, muscle excitation and phospholemman increases the Na+

affinity of all of the NKA heterodimers.