1.10.1. Identification of hSPCA1 & its Splice Variants
Human SPCA1 (hSPCA1) was originally discovered in two studies that focused on the genetic cause of Hailey-Hailey disease (HHD) (to be discussed later) (Hu et al., 2000, Sudbrak et al., 2000). Its gene, named ATP2C1, is located at chromosome 3 in humans at position 3q21 and is the loci for mutations associated with HHD (Ikeda et al., 1994). The ATP2C1 gene sequence matches considerably well with those that encode the P-type Ca2+-ATPases rat SPCA (97%) and yeast PMR1 (49%) (Sudbrak et al., 2000). Four open reading frames can be found between exon 26 and 28 of its gene sequence (Hu et al., 2000, Sudbrak et al., 2000). Only two alternatively spliced
mRNA sequences from ATP2C1 were initially found, which were named ATP2C1a and ATP2C1b (Hu et al., 2000, Sudbrak et al., 2000). This was followed later on by the isolation of two more splice variants, which were named ATP2C1c and ATP2C1d (Fairclough et al., 2003).
So far, four hSPCA1 protein splice variants have been identified and are named hSPCA1 -a, -b, -c and -d. It has been possible to express all except the
hSPCA1c splice variant in HEK 293 cells. Sequence analysis of hSPCA1c shows this variant does not have a part of its amino acid sequence that corresponds to a complete transmembrane helix M10 structure, which is found in the other three splice variants. Furthermore, attempts to measure Ca2+-stimulated thapsigargin-insensitive phospho- enzyme formation and Ca2+ transport activity from this variant have been
unsuccessful, unlike the other three variants (Dode et al., 2005). Taken together, it has been suggested that hSPCA1c is non-functional.
Kinetic parameters associated with Ca2+-ATPase activity of the remaining three hSPCA1 splice variants have been measured and compared to SERCA1a, following their over-expression in COS-1 cells. From this, hSPCA1 functional splice variants have been demonstrated to have lower maximum ATP-binding affinities, slower ATP turnover rates (attributed to their slower rates of dephosphorylation) and higher Ca2+-binding affinities than SERCA1a. The latter would apparently conflict
with rates of Ca2+ dissociation from these Ca2+-ATPases being faster than SERCA1a. However, this was explained by the idea that the apparent higher Ca2+-binding affinities of hSPCA1 -a, -b and -d are not due to stronger binding of Ca2+ but is instead caused by slow processing of the Ca2+-bound phosphoenzyme intermediate. ATP turnover rates of the three functional hSPCA1 isoforms are not affected by pH or K+ concentration, unlike SERCA1a (Dode et al., 2005).
1.10.2. hSPCA2
The isoform of hSPCA1, named hSPCA2, shares 64% amino acid identity with hSPCA1 (Xiang et al., 2005). Unlike hSPCA1, hSPCA2 has a more limited tissue distribution for its expression, whereby the tissue types that have been shown to express the most hSPCA2 are the testes and brain in one study (Xiang et al., 2005), and stomach and rectal tissue in another study (Vanoevelen et al., 2005a). As shown in figure 1.10.2.1, the latter study involved a more extensive search into the tissue distribution pattern of hSPCA2 by using mRNA samples from 83 different tissue types, compared to protein samples from 9 references in the former study, which did not test stomach and rectal tissue. The trans-Golgi network is where hSPCA2 has been observed to localise (at least in rat hippocampus-derived neuronal cells), along with its vesicular derivatives, with a “perinuclear” and “punctate” pattern of
distribution. This is the same as hSPCA1 according to previous observations (Behne et al., 2003), but in contrast to PMR1’s localisation in the medial-Golgi apparatus compartment in yeast (Durr et al., 1998). Sub-cellular localisation of hSPCA2 in human colon tissue also showed it is located primarily at the Golgi apparatus, whilst having a “juxtanuclear” distribution pattern at this organelle with hSPCA1
(Vanoevelen et al., 2005b).
A study using a mutant yeast strain, which failed to express PMR1 but was transformed to express hSPCA2, showed the Mn2+ handling efficiency of hSPCA2 is the same as hSPCA1. However, hSPCA2 does not transport Ca2+ as efficiently as hSPCA1. The lower Ca2+ transport efficiency of hSPCA2 is explained by its lower binding affinity for Ca2+ compared to hSPCA1, which is evident from their Km values
of 1.35µM and 0.25µM, respectively. Furthermore, the lower Ca2+ affinity of hSPCA2 could be due to its EF hand-like motif (a motif commonly associated with
Ca2+-binding) being less well conserved compared to hSPCA1. Yeast cells made to express hSPCA2 in the absence of PMR1 are less efficient in handling unfolded
Figure 1.10.2.1. Tissue Distribution of hSPCA1 & hSPCA2 Expression
(A) Western blots showing the detection of hSPCA2 protein (~100kDa; molecular masses noted on the left) in only brain and testes out of 9 different tissue types (ii). The protein band detected in heart tissue at blot D was present
on the control blot, thus not hSPCA2 protein (i). Lung, kidney and spleen tissues had produced ~100kDa hSPCA2 protein bands in other Western blots
(Xiang et al., 2005). (B) mRNA hybridisation dot blots for hSPCA2 (i) and hSPCA1 (ii) at 83 different tissue types (iii) (Vanoevelen et al., 2005b).
B
(i)
(ii)
(iii)
A
(i)
proteins compared to wildtype PMR1-expressing cells. Taken together, hSPCA2 expression in the brain and its conserved Mn2+ transport efficiency (with loss of Ca2+ transport ability) have been linked to Mn2+ neurotoxicity-related disease, such as Parkinson’s disease, whereby hSPCA2 could contribute to the brain’s ability to deal with Mn2+ toxicity (Xiang et al., 2005).
Over-expression of hSPCA2 in mammalian COS-1 cells has been shown to not affect their endogenous protein levels of hSPCA1 nor SERCA2b. With regards to ion-transporting efficiency, hSPCA2 is more efficient in Mn2+ transport than hSPCA1 and has a Kmfor Mn2+ binding of 0.27µM when over-expressed in COS-1 cells. This
Km value differs from that determined using yeast cells made to express hSPCA2,
which may be due to differing Golgi apparatus structure and lipid composition (Vanoevelen et al., 2005a, Xiang et al., 2005).
1.10.3. The Kinetics of hSPCA2 Activity
One study has compared the functional kinetics of hSPCA2 with that of one of the four hSPCA1 splice variants, hSPCA1d (Dode et al., 2006). From this, hSPCA2 has been shown to have a weaker apparent affinity for Ca2+ but slower rate of Ca2+
dissociation than hSPCA1d. The conflict between these two findings is explained by the E1-P(Ca2+) to E2-P transition being faster for hSPCA2 than hSPCA1d. Another
difference between the two hSPCA isoforms is that hSPCA2 is more prone to thapsigargin inhibition, which is shown by Km values of 28µM for hSPCA1d and
2µM for hSPCA2. This difference in sensitivity to thapsigargin has been attributed to a higher dephosphorylation rate and slower rate of E2 to E1 transition, producing a greater amount of hSPCA2 protein found in the E2 state (which thapsigargin binds to) at any given time compared to hSPCA1. The hSPCA2 isoform also has a higher ATP turnover rate than hSPCA1d, though it was still lower than that for SERCA1a. Furthermore, hSPCA2 has a lower affinity for phosphate binding and consequently a faster dephosphorylation rate compared to both hSPCA1d and SERCA1a, which may be the cause of a faster ATP turnover rate observed for the former Ca2+-ATPase.
The two hSPCA isoforms resemble each other with regard to their lack of sensitivity to pH and K+ concentration. The lack of sensitivity to pH changes, which was recognised by the authors to be characteristic of the hSPCA pumps, has been attributed to these pumps being embedded in Golgi apparatus membranes (Dode et al., 2005). Such membranes have a higher content of sterols and sphingolipids than
ER membranes and this property of the former make them ideal for stabilising the E2 state of a Ca2+-ATPase, which is unstable following the release of its bound Ca2+ because of the exposure of multiple negative charges at the Ca2+-binding site. The E2 state of SERCA is instead stabilised by the binding of protons and filling of the Ca2+- binding site with water molecules, which makes this Ca2+-ATPase sensitive to pH (Michelangeli et al., 1990). The lack of K+ sensitivity by both hSPCA isoforms has been suggested to be due to the absence of an acidic residue corresponding to E732 in SERCA, which has been found to be involved in K+-binding in the latter Ca2+-ATPase (Shigekawa et al., 1978). A neutral (N704 in hSPCA2) or shorter (D674 in hSPCA1d) residue at the equivalent position in the amino acid sequence of hSPCA is found instead.
1.10.4. hSPCA1 & Human Spermatozoa
An example of a cell type that uses hSPCA1 for a specialised role is human
spermatozoa. This cell type has been shown to express hSPCA1, which is likely to contribute to progesterone-stimulated Ca2+ oscillations because it is a process that
involves the mobilisation of Ca2+ from intracellular stores not controlled by SERCA
pumps (shown by their insensitivity to both thapsigargin and CPA inhibition) (Harper et al., 2005). The association of hSPCA1 with such Ca2+ oscillations in human sperm
cells make it unlikely to be involved in the acrosome reaction, which contrasts observations made previously in sea urchin spermatozoa (Gunaratne and Vacquier, 2006). Like yeast, mature human spermatozoa are unlikely to express SERCA-type pumps, which makes hSPCA1 the main intracellular Ca2+-ATPase in this cell type. In human sperm, hSPCA1 is located in the region between the anterior midpiece and the rear of the sperm head (a region known as the redundant nuclear envelope) (Harper et al., 2005), unlike SPCA’s mitochondrial localisation in sea urchin sperm (Gunaratne and Vacquier, 2006).