Structure and function of NKA

The Na+_,K+_{-ATPase (NKA) is a P-type ATPase heterodimer protein consisting of an α and β} subunit (some tissues express also express a γ subunit), which translocates 3 Na+ _{ions out of} the cell and 2 K+ _{ions into the cell per molecule of ATP hydrolysed. The α subunit is referred} to as the catalytic subunit where both the Na+ _{and K}+ _{are exchanged and ATP is hydrolysed.} The β subunit has a regulatory role in modulating NKA activity and is also implicated in mobilisation and routing of NKA into the cell membrane (Clausen, 2003). The NKA also has a site for binding of cardiac glycosides, presumably for endogenous digitalis-like compounds (Hamlyn et al., 1996; Crambert et al., 2002), which can also be targeted pharmacologically (Gheorghiade et al., 1997; Clausen, 2003). The structure of NKA is described in Figure 2.6.

Figure 2.6: The molecular structure Na+_,K+_{-ATPase (NKA), including phospholemman} (FXDY1). Published originally by Clausen (2013), created by Flemming Cornelius (Aarhus University).

2.3.4.1 Subunits and isoforms of NKA

The catalytic α subunit of NKA constitutes the majority of the NKA heterodimer (Figure 2.6), at a molecular weight of 112 kDa. The process of how NKA exchanges Na+ _{and K}+ _{is well}

established (Post et al., 1972; Glynn, 1993). Before activation, NKA is accessible to the interior of the cell and have a higher affinity for Na+ _{ions, which with ATP present causes}

3Na+ _{to be trapped in the NKA. The energy produced from the hydrolysis of an ATP then}

causes a conformational change in the shape of the protein. After this conformational change, the NKA is now accessible to the extracellular space and the pump loses its affinity

for Na+_{, causing the release of Na}+ _{ions into the extracellular space. When the NKA is}

accessible to the extracellular space, it has a higher affinity for K+ _{ions, causing 2K}+ _{ions to}

become trapped in the protein. The NKA then undergoes another conformational change reversing then to be accessible to the cell interior, then releasing the 2K+ _{ions into the cell}

and returning the NKA to its original state. Figure 2.7 is a simplified diagram of the above procedure as described by Glynn (1993).

Figure 2.7: Schematic of the processes involved in Na+ _{and K}+ _{exchange in the Na}+_,K+_-

ATPase (NKA) (Glynn, 1993).

While the β subunit (~38 kDa) of NKA is not directly involved in the process of Na+ _{and K}+

occlusion and exchange, it does have a vital regulatory role in NKA and other P-type ATPases. The β subunit of NKA is responsible for the transport of the maturing NKA to and

insertion into the cell membrane and plays a role in preventing cellular degradation of the NKA (Geering, 2001). The correct routing and unfolding into the cell membrane seems to influence the enzymatic properties of the α unit (Hasler et al., 1998; Geering, 2001; Kristensen et al., 2010). The γ subunit, or FXDY accessory protein(s) is present in most tissues; and is associated with activation of NKA (Béguin et al., 1997). In skeletal muscle, FXYD1 ( phospholemman) has an important regulatory role on NKA activity (Crambert et al., 2002; Crambert et al., 2003).

Both α and β subunits of NKA have several distinct isoforms with four α isoforms (α1, α2, α3

and α4) and three β isoforms: (β1, β2 and β3) known to exist in mammalian tissues (Blanco et

al., 1998). The α isoforms are expressed tissue-specifically, with each isoform seeming to have a tissue specific function (Blanco et al., 1998; Kristensen et al., 2010). In skeletal muscle, the existence of α1 and α2 has been conclusively shown in humans (Hundal et al.,

1994; Murphy et al., 2008a; Kristensen et al., 2010) and in rats (Fowles et al., 2004). The α3

isoform is found in low abundance in human skeletal muscle (Hundal et al., 1994; Murphy et al., 2004; Kristensen et al., 2010). It should be noted that the α4 isoform is expressed

primarily in the testes (Shamraj et al., 1994; Lingrel et al., 2003) and sperm (Hlivko et al., 2006), although some evidence suggests it does exist in human muscle (Keryanov et al., 2002). Further discussion on the α4 isoform is beyond the scope of this review due to its

extremely low, if any, content in skeletal muscle.

The β1, β2 and β3 isoforms of NKA are all present in skeletal muscle in rats (Arystarkhova et

al., 1997; Fowles et al., 2004). Whilst β1 and β2 are consistently reported in human skeletal

detect the expression of the β2 isoform (Hundal et al., 1994; Juel et al., 2000a; Kristensen et

al., 2010). This has been refuted though by several studies which have reported the expression and content of both the β2 and β3 isoform in human skeletal muscle (Murphy et

al., 2004; Murphy et al., 2008a). The family of FXYD (1-7) proteins is expressed tissue specifically when associated with the NKA. In skeletal muscle FXDY1 (phospholemman; PLM) is the only FXDY protein present and is an important acute activator of NKA during muscle contraction.

The function of each individual isoform is poorly understood. In Xenpus oocytes, a cell from a genus of frog, all of the isoform combinations which were expressed were found to be functional (Crambert et al., 2000). The α1 subunit has traditionally been thought to play a

‘housekeeping’ role, due to its location primarily in the sarcolemma and its higher affinity for both Na+ _{and K}+ _{compared to the α}

2 isoform during resting conditions (Crambert et al.,

2000). Further, the α1 isoform is the most abundant isoform in cardiac muscle, whereas the

α2 isoform is the most abundant in skeletal muscle (Orlowski et al., 1988; Hansen, 2001).

However, partial knockout of α1 in mice decreased muscle contractile strength (Lingrel et

al., 2003), indicating some role in muscle contraction. Conversely, the α2 isoform makes up

~75-85% of NKA in rat EDL muscle (Hansen, 2001) and muscle NKA content is increased by chronic physical exercise in humans and rodents (Kjeldsen et al., 1986; Green et al., 1993; McKenna et al., 1993; Green et al., 1999a). Research using mice confirms the importance of the α2 isoform for exercise and contractile force. Partial global knockout which caused a

50% reduction in α2 impaired contractile force in mouse isolated muscle (Lingrel et al.,

strength, endurance and exercise tolerance (Radzyukevich et al., 2013). In mice with complete global knockout of either α1 or α2 isoform, the mice had either invalid formation

of the embryo or death moments after birth (Lingrel et al., 2003). The role of the α3 isoform

is unknown in skeletal muscle.

The function of the individual β isoforms with NKA is not clear. The β1 isoform tends to have

a higher affinity to Na+ _{compared to β}

2, regardless of the α isoform of the NKA (Crambert et

al., 2000), suggesting that like α1, the β1 may be primarily utilised in basal conditions. It

should be noted however that the above differences in Na+ _{and K}+ _{affinity with NKA}

isoforms are altered by muscle contraction/exercise, likely due to the phosphorylation of PLM (Juel, 2009; Cirri et al., 2011; Juel et al., 2013). Hence, investigating the function of each NKA isoform via Na+ _{affinity alone without the presence or phosphorylation of PLM is}

not appropriate in vivo. Overall, there is very limited evidence conclusively demonstrating the separate functions of the β NKA isoforms in skeletal muscle.