As mentioned above, the SSM technique was used to investigate net charge translocation (electrogenic transport) in P-type ATPases. Charge displacement associated with specific steps, i.e., ion binding/release, ion translocation, and exchange was measured in the ATPase transport cycle and the electrogenicity of partial reactions was determined, thereby providing mechanistic insights in the transport mechanism of different P-type ATPases. For example, a direct proof for the electrogenicity of cytoplasmic Na
+ binding to the Na
+,K
+-ATPase was obtained with Na
+ concentration jump experiments performed on membrane fragments containing Na
+,K
+-ATPase adsorbed on the SSM [
26]. It was found that the charge associated with the Na
+ binding step is about 30% of the displaced charge related to Na
+ translocation and release, indicating that cytoplasmic Na
+ binding is a minor electrogenic event in the reaction cycle of Na
+,K
+-ATPase [
26].
In the next sections, we will discuss the contribution of the SSM technique to unravel key features of the electrogenic transport activity of some prominent members of the P-type ATPase family. In particular, the focus of the present review is on SERCA, Cu+-ATPases ATP7A and ATP7B, and P4-ATPase (phospholipid flippase) ATP8A2. SERCA has been characterized in detail by the SSM technique, providing useful information on the enzyme’s transport mechanism. This information was used for a comparative analysis of the transport properties of the Cu+-ATPases and phospholipid flippase, which were recently investigated by the SSM method.
3.1. Sarcoplasmic Reticulum Ca2+-ATPase
The SERCA enzyme is one of the most investigated P-type ATPase [
15,
16,
37,
38,
39]. In muscle cells, SERCA couples the energy gained by the hydrolysis of one ATP molecule to the transport of two Ca
2+ ions against their electrochemical potential gradient from the cytoplasm into the lumen of SR, which is the main intracellular Ca
2+ storage organelle. Ca
2+ uptake in the SR lumen by SERCA plays an essential role in regulating cytoplasmic Ca
2+ concentration, which is kept at or below 0.1 µM; in this manner, SERCA induces muscle relaxation and contributes to intracellular Ca
2+ homeostasis. Modified SERCA expression and impaired pumping activity have been associated with pathological conditions and several diseases with a wide range of severity [
39,
40].
SERCA (approximately 110 KDa) belongs to the P2A-ATPase subfamily. In mammals, SERCA is encoded by three different genes, ATP2A1-3, but isoform diversity is increased by alternative splicing of the transcripts, which raises the number of possible SERCA isoforms to more than 10 [
41,
42]. A very convenient experimental system for functional and structural studies of SERCA is provided by vesicular fragments of longitudinal SR, where SERCA1a is the predominant isoform. SR vesicles contain a high amount of SERCA, which accounts for approximately 50% of the total protein and which reaches a density in the SR membrane of about 30,000 µm
−2 [
43].
Electrical currents generated by SERCA were measured by adsorbing native SR vesicles containing SERCA1a from rabbit skeletal muscle on the SSM and by activating the calcium pumps with substrate, i.e., Ca
2+ and ATP concentration jumps. The observed current signals allow the direct measurement of charge translocation by SERCA under different activation conditions. In particular, charge movements related to different electrogenic partial reactions in the SERCA transport cycle were detected. It was shown that a Ca
2+ concentration jump in the absence of ATP induces a transient current (dotted line in
Figure 3A), which is associated with an electrogenic event corresponding to enzyme activation by the initial binding of Ca
2+ to the cytoplasmic side of the ATPase (the exterior of the SR vesicle, see
Figure 2) [
27,
28,
44]. When an ATP concentration jump was performed in the presence of Ca
2+ ions, a current signal was detected (solid line in
Figure 3A), which is associated with a further electrogenic step corresponding to ATP-dependent calcium translocation by the enzyme [
20,
27]. In particular, ATP concentration jump experiments on SR vesicles in the presence and absence of a calcium ionophore at different pH values [
27] indicated that the ATP-induced electrical current is related to displacement and release of pre-bound Ca
2+ at the luminal side of the pump (the interior of the SR vesicle, see
Figure 2) after phosphorylation of the enzyme by ATP. The transient currents measured after a Ca
2+ jump in the absence of ATP and an ATP jump in the presence of Ca
2+ were both fully suppressed by thapsigargin [
44], which is a highly specific and potent SERCA inhibitor [
45,
46]. We point out that to perform ATP hydrolysis and active Ca
2+ transport SERCA undergoes large domain movements enabled by dynamic fluctuations and conformational transitions that are not random but instead are driven by the availability of specific substrates [
47].
It is interesting to observe that the amplitude of the signal related to ATP-dependent Ca
2+ translocation decreases as the pH is raised from 7 to 8 (
Figure 3B). It is known that exchange of Ca
2+ with H
+ is a specific feature of SERCA [
37,
48], which favors Ca
2+ release at the luminal side [
17,
49]. Useful information was provided by previous measurements on reconstituted proteoliposomes containing SERCA [
49,
50,
51,
52]. In particular, it was shown that the stoichiometry of the Ca
2+/H
+ countertransport is about 1/1 when the luminal and medium pH is near neutrality [
49,
52]. The importance of Ca
2+/H
+ exchange in determining the release of bound Ca
2+ from the phosphoenzyme E
2P was demonstrated in steady-state experiments on native SR vesicles [
37]. It was reported that the maximal levels of accumulated Ca
2+ are significantly reduced if the pH is raised above 7. This result shows that if exchange is limited due to low H
+ concentration, Ca
2+ is less likely to dissociate from the phosphoenzyme. Thus, the pH dependence of the current signals obtained with ATP concentration jumps (inset of
Figure 3B) also indicates that when a lack of H
+ limits Ca
2+/H
+ exchange, i.e., alkaline pH, the translocation of bound Ca
2+ is prevented, even though K
+ is present in high concentration and may neutralize acid residues at alkaline pH [
48]. This suggests a requirement for specific H
+ binding at the Ca
2+ transport sites in order to obtain Ca
2+ release.
The SSM method has also been used to investigate a very interesting research topic, which is currently receiving much attention, i.e., the molecular mechanisms of SERCA regulation. In muscle cells, SERCA transport activity is regulated by two analogous transmembrane proteins: phospholamban (PLN, 52 amino acids), which is primarily expressed in cardiac muscle where it regulates the SERCA2a isoform [
53], and sarcolipin (SLN, 31 amino acids), which is mainly expressed in skeletal muscle where it regulates the SERCA1a isoform [
54]. In particular, PLN inhibits pump activity by lowering the apparent Ca
2+ affinity of SERCA, and the phosphorylation of PLN by protein kinases relieves SERCA inhibition [
53]. There is general consensus that the PLN inhibition of SERCA involves the reversible physical interaction of a PLN monomer under calcium-free conditions. However, experimental evidence was provided that a PLN pentamer, which has been described as an inactive storage form, can also interact with SERCA [
55,
56].
To investigate the PLN effect on ATP-dependent Ca
2+ translocation by SERCA, SSM-based current measurements were carried out on co-reconstituted proteoliposomes containing SERCA and PLN [
57]. The proteoliposomes were adsorbed on the SSM and activated by Ca
2+ and/or ATP concentration jumps. In particular, substrate conditions (various Ca
2+ and ATP concentrations) were chosen that promoted specific conformational states of SERCA, from which calcium transport could be initiated. The results from pre-steady state charge (calcium) translocation experiments were compared with steady-state measurements of ATPase hydrolytic activity. It was found that the PLN effect on SERCA transport activity depends on substrate conditions, and PLN can establish an inhibitory interaction with multiple conformational states of SERCA (a calcium-free E
2 state, a E
1-like state promoted by Ca
2+, and a E
2-like state promoted by ATP, shown in red in
Figure 4) with distinct effects on SERCA’s kinetic properties [
57]. It was also noted that once a particular SERCA–PLN inhibitory interaction is established, it remains throughout the SERCA transport and catalytic cycle. These findings were interpreted on the basis of a conformational memory [
58,
59] in the interaction of PLN with SERCA, whereby a defined structural state of the SERCA/PLN regulatory complex, which depends on substrate conditions, is retained during SERCA turnover and conformational cycling.
In addition to PLN and SLN, single-span transmembrane proteins have recently been discovered that act as regulators of SERCA activity: dwarf open reading frame (DWORF), myoregulin (MLN), endoregulin (ELN), and another-regulin (ALN) [
60,
61,
62]. While MLN, ELN, and ALN have been identified as inhibitors of SERCA activity, it was shown that DWORF does not inhibit the SERCA pump [
62], enhancing Ca
2+ uptake by displacing PLN. The oligomerization of these new SERCA regulators and the binding interaction of the monomeric form with the calcium pump were very recently investigated [
63], thus providing a useful contribution in the characterization of the complexity of SERCA regulatory mechanisms. In this respect, it appears that the above-mentioned transmembrane peptides could be conveniently investigated by the SSM technique upon their reconstitution in proteoliposomes containing SERCA. This would help to elucidate the inhibitory or activation effects of the recently discovered SERCA regulators.
3.2. Cu+-ATPases ATP7A and ATP7B
The mammalian copper ATPases ATP7A and ATP7B are 165–170 KDa membrane proteins belonging to subclass IB of the P-type ATPase superfamily. At normal copper levels in the cell, ATP7A and ATP7B are found in the trans-Golgi network (TGN), and these enzymes translocate copper across the membrane from the cytoplasm into the TGN lumen using ATP hydrolysis [
64,
65,
66,
67,
68]. ATP7A and ATP7B contribute to intracellular copper homeostasis by delivering copper to newly synthesized copper-containing proteins in the TGN and by removing copper excess from the cell [
64]. ATP7A is expressed in most tissues but not in the liver, whereas ATP7B is mainly found in this organ [
64]. The malfunction of either ATP7A or ATP7B is the cause of severe diseases, which are known as Menkes (ATP7A) and Wilson (ATP7B) diseases.
ATP7A and ATP7B show high sequence homology (about 60% identity). Their structure comprises eight transmembrane helices, which include a copper binding site (transmembrane metal binding site, TMBS), and the A, N and P cytoplasmic domains, which are common for P-type ATPases. A unique structural feature of ATP7A and ATP7B is the highly mobile N-terminal chain of six copper binding domains (N-terminal metal binding domain) that are involved in the copper-dependent regulation and intracellular localization of these enzymes [
69].
As described in several reviews (e.g., [
64,
65,
70,
71,
72,
73,
74]), Cu
+ transfer by ATP7A and ATP7B involves copper acquisition from donor proteins on the cytoplasmic side of the membrane and copper delivery to acceptor proteins on the luminal side, without establishing a free Cu
+ gradient. In conformity with other P-type ATPases, ATP7A and ATP7B hydrolyze ATP to form a transient phosphorylated intermediate, and they undergo conformational transitions that favor Cu
+ transfer to/from the TMBS. From high-resolution crystal structures and molecular dynamics simulations on a bacterial Cu
+-ATPase (
Legionella pneumophila Cu
+-ATPase, LpCopA) [
75,
76], it appears that copper ATPases have a unique copper release mechanism that is likely to be involved in specific and controlled Cu
+ delivery to acceptor proteins.
Electrogenic copper movement within mammalian copper ATPases was demonstrated by current measurements on COS-1 microsomes expressing recombinant Cu
+-ATPases (ATP7A and ATP7B) adsorbed on an SSM [
31,
48]. When an ATP concentration jump was performed on microsomes containing ATP7B (or ATP7A) in the presence of CuCl
2 and dithiothreitol to reduce Cu
2+ to Cu
+, a current signal was obtained (solid line in
Figure 5A), which was not observed when bathocuproinedisulfonate (BCS), acting as Cu
+ chelator, was added to the reaction buffer (dotted line in
Figure 5A). These experiments indicate that the copper-related current signal is associated with an electrogenic event corresponding to Cu
+ movement within ATP7B upon phosphorylation by ATP [
31,
48], which is consistent with copper displacement from the TMBS to the luminal side of the enzyme.
By fitting the decay phase of the transient current with a first-order exponential decay function, a charge transfer decay time constant (τ) of 140 ms was determined for ATP7B, which is within the time frame of aspartate phosphorylation by ATP [
31], suggesting that copper displacement in ATP7B is correlated to formation of the phosphorylated intermediate and precedes phosphoenzyme hydrolytic cleavage. This conclusion was also supported by SSM-based current measurements on the D1044A mutant of ATP7A. Asp1044 is the conserved aspartate residue in the P-domain of ATP7A that interacts with ATP to form the aspartyl phosphorylated intermediate. It was shown that the D1044A mutant yielded no current signal upon an ATP concentration jump in the presence of Cu
+ [
77]. This result further indicated that ATP-dependent copper movement through the ATPase is directly correlated to formation of the aspartyl phosphorylated intermediate by ATP consumption.
It is interesting to observe that ATP-induced copper movement in mammalian Cu
+-ATPases is significantly slower than ATP-dependent Ca
2+ translocation in SERCA [
31], as shown by the different decay time constants τ for charge displacement following ATP jumps (inset of
Figure 5A) on ATP7B (red line, τ = 140 ms) and SERCA (black line, τ = 25 ms). It is worth mentioning that the τ values for charge movements in ATP7B and SERCA are consistent with a slower phosphoenzyme formation in the copper ATPase [
31] with respect to SERCA [
78]. It should be noted that these decay time constants are attributed to initial partial reactions of the pump transport cycle and are not equivalent to steady-state turnover [
31].
SSM measurements on ATP7A and ATP7B revealed that ATP-induced charge movement in these enzymes is not changed by alkaline or acid pH [
48], as shown by charge transfer measurements at different pH values (
Figure 5B). This finding indicated that copper displacement in ATP7A and ATP7B is pH independent, and it highlights a significant difference in the transport mechanisms of ATP7A/B and SERCA. It was proposed that in ATP7A/B, Cu
+/H
+ exchange may not be required for luminal copper release [
48], as opposed to the strict requirement of Ca
2+/H
+ exchange in SERCA as discussed above. It is worth mentioning that carboxylate residues are absent in the ion-binding cluster located in the transmembrane region of the bacterial
Archaeoglobus fulgidus CopA [
79] and LpCopA [
30], while crucial aspartate and glutamate residues are present in the equivalent transmembrane domain of SERCA [
12,
16,
80] that are directly involved in Ca
2+/H
+ exchange. Thus, SSM measurements on ATP7A/B supported the hypothesis that Cu
+ release in these enzymes may not be coupled to a net proton countertransport, which has not been observed for PIB-type ATPases [
72,
73,
81]. Interestingly, a very recent study reported real-time fluorescence measurements on
E.coli Cu
+-ATPase (EcCopA) reconstituted in small unilamellar vesicles encapsulating a set of fluorescence probes that are selective for Cu
+, pH, and membrane potential [
82]. The results of this study demonstrated the absence of H
+ countertransport in the Cu
+ translocation cycle of EcCopA, qualifying EcCopA as an electrogenic uniporter.
3.3. P4-ATPase ATP8A2
A characteristic feature of eukaryotic cell membranes is the asymmetrical distribution of different lipids across the membrane bilayer. This is particularly evident in the plasma membrane, where phosphatidylcholine (PC) and sphingolipids, i.e., sphingomyelin and glycosphingolipids, are concentrated in the exoplasmic leaflet of the membrane, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) are mainly restricted in the cytoplasmic leaflet [
83,
84,
85,
86]. Membrane lipid asymmetry is essential for a variety of cellular processes that include, e.g., cell and organelle shape determination, membrane stability and impermeability, membrane curvature, vesicle formation and trafficking, host–virus interactions, membrane protein regulation, blood coagulation, and apoptosis [
86,
87,
88,
89,
90].
Phospholipid flippases, belonging to the P4-ATPase subfamily, couple ATP hydrolysis to the translocation of specific phospholipids from the exoplasmic to the cytoplasmic leaflet of biological membranes in order to generate and maintain transmembrane lipid asymmetry [
89,
91,
92,
93,
94,
95]. P4-ATPases are only found in eukaryotes and constitute the largest P-type ATPase subfamily. In mammals, at least 14 P4-ATPases are known, which are divided into five classes [
89]. P4-ATPase dysfunction has been associated with severe neurological disorders and liver diseases in humans [
92]. These lipid transporters consist of a large polypeptide with a molecular mass of about 120 kDa, which shares the general architecture of P-type ATPases. Most P4-ATPases form a heterodimeric complex with an accessory β-subunit of about 50 kDa belonging to the CDC50/LEM3 family [
89,
96,
97]. High-resolution structures of yeast [
98,
99] and human [
100] lipid flippases were determined by cryo-electron microscopy, as reviewed in [
101], and very recently, the crystal structures of a human plasma membrane flippase were also reported [
102].
The transport mechanism of P4-ATPases is the subject of intensive research, and various models have been proposed for the phospholipid translocation pathway in P4-ATPases [
103,
104,
105,
106,
107]. The recent atomic resolution structures of yeast and human P4-ATPases have provided valuable information on different conformational states in the flippase transport cycle, which is depicted by the Albers–Post or E
1–E
2 scheme commonly used to describe the mechanism of ion transporting P2-type ATPases. The P4-ATPase reaction cycle (see the simplified diagram in
Figure 6A) has been examined in some detail for the mammalian flippase ATP8A2 [
108], which is highly expressed in the retina, brain, testis, and spinal cord. It was shown that ATP8A2 is phosphorylated by ATP at the aspartate conserved in all P-type ATPases, and the phosphoenzyme exists in E
1P and E
2P states [
108]. Dephosphorylation of the E
2P state is activated by binding of the two known substrates PS and PE, but not by binding of PC that is not a substrate of ATP8A2 [
109], and dephosphorylation is associated with lipid translocation from the exoplasmic to the cytoplasmic leaflet of the membrane bilayer. Although significant progress has been made in our understanding of phospholipid flipping by P4-ATPases, several aspects of the flippase transport mechanism remain to be explored, such as the stoichiometry of phospholipid molecules translocated per ATP hydrolyzed, the mechanisms underlying lipid binding and release, the electrogenicity of phospholipid transport, and the related issue of countertransport, i.e., countertransport of an ion or other charged substrate from the cytoplasm to the exoplasm in connection with the E
1 → E
1P → E
2P reaction sequence as observed for P2-type ATPases.
To address unexplored key aspects of the flipping mechanism of P4-ATPases, in particular the electrogenicity of phospholipid flippases and ion countertransport, the SSM method was very recently used in a study of the elctrogenic properties of wild-type and mutant forms of the flippase ATP8A2 [
32]. Purified ATP8A2 and its accessory CDC50A protein were reconstituted in proteoliposomes of different lipid compositions that were adsorbed on the SSM surface and subjected to ATP concentration jumps. It was shown that an ATP jump on ATP8A2 reconstituted into proteoliposomes consisting of a mixture of 90% PC and 10% PS induced a current signal (black line in
Figure 6B) that was completely suppressed in the presence of the ATPase inhibitor orthovanadate (red line
Figure 6B). Since orthovanadate binds to the ATPase from the cytoplasmic side, it was concluded that the ATPase molecules with the cytoplasmic side facing the external aqueous solution generated the ATP-dependent charge movement across ATP8A2. It was also noted that the sign of the ATP8A2-related current signal is positive, as observed for the SERCA-related transient current (inset of
Figure 6B) that is attributed to the translocation and release of Ca
2+ ions into the SR vesicle interior [
27] (see
Section 3.1). We point out that the movement of positive charge in one direction is electrically equivalent to the displacement of negative charge in the opposite direction. Therefore, the ATP8A2 current signal was associated with ATP-dependent translocation of the negatively charged PS toward the outside of the proteoliposomes (the ATP8A2 cytoplasmic side facing the external aqueous solution) [
32].
It is worth noting that PC, also present in the proteoliposomes, is not a substrate for ATP8A2 and has an electrically neutral head group. As mentioned above, PC does not stimulate ATP8A2 dephosphorylation; however, the enzyme can be phosphorylated by ATP in the absence of PS and PE and in the presence of PC alone [
108]. Interestingly, an ATP concentration jump on ATP8A2 reconstituted in proteoliposomes containing 100% PC yielded no significant transient current, indicating that enzyme phosphorylation by ATP (E
1→E
1P reaction step) does not generate any electrical signal [
32].
Useful information was also provided by ATP concentration jump experiments on proteoliposomes containing selected mutants of ATP8A2 [
32]. In particular, the E198Q mutation was examined. Glu-198 is located in the DGET motif of the cytoplasmic A-domain of ATP8A2 that facilitates dephosphorylation of the phosphorylated intermediate. It was reported that phosphorylation by ATP is allowed in E198Q, while dephosphorylation is blocked with resulting E
2P accumulation [
108]. The absence of an electrical current upon an ATP concentration jump on proteoliposomes containing E198Q indicated that the electrogenicity of ATP8A2 is not related to the E
1→E
1P step (phosphorylation by ATP) or with the E
1P→E
2P conformational transition of the enzyme, which is in agreement with the experiments on 100% PC proteoliposomes. This finding is important to address the issue of whether ion countertransport occurs from the cytoplasmic to the exoplasmic side of the phospholipid flippase. In fact, it was shown that no charged substrate is countertransported during the E
1 → E
1P → E
2P reaction sequence [
32], thereby distinguishing the P4-ATPase from the P2-type ATPases, which transport ions in opposite directions and are therefore referred to as antiporters.
It was concluded that the electrogenicity of ATP8A2 is related to a step in the ATPase transport cycle directly involved in translocation of the phospholipid [
32]: dephosphorylation of the E
2P intermediate, activated by lipid binding from the exoplasmic side, and/or the subsequent E
2 → E
1 conformational transition of the dephosphoenzyme, which is associated with release of the lipid to the cytoplasmic side [
104]. It is noteworthy that the findings of the SSM study of the mammalian phospholipid flippase [
32] support the view that the P4-ATPase is most likely an electrogenic uniporter, as also recently reported for a bacterial Cu
+-transporting P1B-ATPase [
82].