COSA JUZGADA JUDICIAL VERSUS COSA JUZGADA CONSTITUCIONAL: OTRA

4.1.2.1 Identification of two-pore channels as novel members of the voltage-gated cation channel superfamily

In 2000 the two-pore channel subtype 1 (TPC1; gene name TPCN1) was first cloned and identified as a novel two-domain channel (Ishibashi et al., 2000). The protein, deduced from screening the rat kidney cDNA library, was reported as being 819 amino acid residues with a calculated molecular weight of 94.6 kDa (Ishibashi et al., 2000). Mammalian TPCs are comprised of 12 transmembrane (TM) α-helices

containing 2 homologous domains of 6 TM α-helices (S1-S6) and a re-entrant P-loop

incorporated between S5 and S6 of each domain (Fig. 4.1A). Their characteristic structure indicates that TPCs are new members of a family of proteins belonging to the superfamily of voltage-gated cation channels, which is composed of over 140 members that are related by both structural and functional motifs (Yu et al., 2005). The P-loop is one of the important functional elements of this ion channel superfamily and is thought to sit within the pore and contribute towards ion conductance and ion selectivity (Yu et al., 2005). As previously stated, the pore-forming subunits of voltage-gated sodium (Nav) and calcium (Cav) channels have 4 domains whereas

proteins are therefore thought to have undergone two rounds of duplication in order to develop the necessary number of domains required to provide the pore region consistent with that conferred to Nav and Cav channels. This led Ishibashi et al. to

suggest that TPCs could be the intermediate product of the 2 rounds of duplication (Ishibashi et al., 2000). Therefore, their structure would likely suggest that TPCs form dimers with each subunit contributing 2 P-loops. Thus upon the formation of a dimer, 4 P-loops come together to form a functional pore (Fig. 4.1A).

Fig. 4.1. Two-pore channels share structural similarity with single-pore and four-pore domain channels and exhibit a wide tissue distribution

A Comparison of the phylogenetic relationship between TPC subtypes and both single-pore and four- pore domain channels suggesting that TPCs may form an intermediate stage between the two rounds of duplication that separate these two gene families. Abbreviations used: at, Arabidopsis thaliana; CaV,

voltage-gated Ca2+ _{channel; d, dog; h, human; Na}

V, voltage-gated Na+ channel; r, rat. B Northern blot

analysis of TPC2 expression in human tissues showing that TPC2 exhibits a wide tissue distribution with abundant expression in liver and kidney in particular. This figure was kindly provided by Mike Zhu.

The structural similarity of TPC1 to Cav channels led Ishibashi et al. to

postulate that TPC1 would be a membrane bound, voltage-gated cation channel (Ishibashi et al., 2000). However, xenopus oocytes injected with TPC1 cRNA

showed no significant difference in membrane currents from the background currents recorded in control cells. Also, isotopic studies with the same cells failed to show any significant Na+ or Ca2+ uptake from the extracellular media. Furthermore Chinese hamster ovary cells (CHOs) expressing TPC1 also failed to demonstrate membrane currents significantly above background (Ishibashi et al., 2000). These data led the authors to suggest that TPCs may need additional proteins for either effective processing and delivery to the membrane, or for the formation of a functional pore. A further suggestion by Ishibashi et al. was that TPCs may require something other than voltage for appropriate gating of the pore. Lastly, the authors suggested that the cellular distribution of the TPC1 protein must be determined in order to establish whether or not TPC1 is targeted to membranes other than the plasma membrane (Ishibashi et al., 2000). This point is most pertinent to the question of the molecular identity of the NAADP receptor, as this would reasonably be expected to be present on the membranes of lysosome-related organelles.

Following the identification of TPC1 in the rat, plant TPC1 was first identified and cloned from Arabidopsis and was termed AtTPC1 (Furuichi et al., 2001). The AtTPC1 protein is 733 amino acid residues in length with an estimated molecular weight of approximately 85 kDa and, similar to rat TPC1 has a predicted 12 TM structure divided into two domains of 6 TM regions each (Furuichi et al., 2001). AtTPC1 expression rescued the Ca2+ uptake activity in the yeast mutant cch1 (Furuichi et al., 2001) and also, sugar-induced luminescence, acting as an indicator for increased cytoplasmic Ca2+ in Arabidopsis leaves, was enhanced by over expression of AtTPC1 and was suppressed by expression of antisense AtTPC1 (Furuichi et al., 2001). In 2005, Sanders and colleagues used a GFP-TPC1 fusion protein to show that AtTPC1 is expressed on membranes distinct from the plasma membrane, and is associated with the vacuolar membrane (Peiter et al., 2005). Using TPC1 over-expressing cells they then determined that AtTPC1 has a role in the development of the cytoplasmic Ca2+-dependent slow vacuolar channel current, which is responsible for Ca2+-dependent Ca2+-release from intracellular vacuoles of plant cells (Peiter et al., 2005). Further confirmation of plant TPC1 functioning as a cation channel present on the vacuolar membrane was provided by Dietrich and colleagues, though the authors suggest that plant TPC1 is a vacuolar Ca2+-gated Ca2+ release channel (Kawano et al., 2004; Ranf et al., 2008). That plant TPC1 may be activated in response to Ca2+ is not inconsistent with its structure, as a notable difference

between plant TPC1 and mammalian TPCs is the presence of EF-hands on plant TPC1 (Ranf et al., 2008). The ability of EF-hands to bind Ca2+ would thus provide plant TPC1 with a degree of Ca2+-sensitivity. Therefore, investigations using plant cell types have shown that plant TPC1 is involved in intracellular Ca2+ signalling and, of particular importance to the present study, that plant TPC1 is located on the membrane of vacuoles rather than the plasma membrane (Fig. 4.2) (Furuichi et al., 2001; Kawano et al., 2004; Peiter et al., 2005; Ranf et al., 2008).

Fig. 4.2. Schematic representation of the major Ca2+ _{signalling pathways in an Arabidopsis} thaliana cell

Depicted are some of the mechanisms by which Ca2+ _{is able cross the plasma membrane and some of}

the major Ca2+ storing organelles including the central vacuole, plastids and the ER. Ca2+ is sequestered into organelles or removed across the cell membrane by autoinhibited Ca2+ ATPases (Pacaud et al.). Ca2+ _{influx may occur via depolarisation-activated Ca}2+ _{channels (DACC), hyperpolarisation-activated}

Ca2+ _{channels (HACC), and voltage-independent Ca}2+ _{channels (VICC). The ER also sequesters Ca}2+

via the ER-type Ca2+ _{ATPase (ECA) and may be mobilised via activation of RyRs and IP}

3Rs. The

large Ca2+_{-storing central vacuole sequesters Ca}2+ _{via ACA and the action of the Ca}2+_/H+ _antiporter

(CAX) coupled to a V-type ATPase. Ca2+ _{may be mobilised from the vacuole via the activation of}

IP3Rs, RyRs, HACCs, the slow-activating vacuolar channel (SV) and the plant two-pore channel

4.1.2.2 Two-pore channels are expressed in a broad range of species and exhibit an extensive mammalian tissue distribution

3 subtypes of TPC have been identified in the animal kingdom (TPC1, TPC2 and TPC3) and one subtype in plants (plant TPC1; also known as AtTPC1). However, it is important to note that plant TPC1 shows low sequence homology to either animal TPC1, TPC2 or TPC3, and is considered to be a plant counterpart to all animal TPCs and not exclusively comparable to animal TPC1. That plant TPC1 is distantly related to all 3 animal subtypes is exemplified by comparison of the conserved TM regions with ≤ 30 % amino acid sequence homology shared with the 3

animal TPC subtypes, which are also equally distant from each other. Fig. 4.3 shows the phylogenetic tree generated from all known TPC sequences. This shows that all 3 TPCs are present in sea urchins and most vertebrates. However, TPC3 appears to be absent in primates and some rodents (e.g. mice and rats; Fig. 4.3). Such a broad species distribution including both animal and plant kingdoms suggests that the genes encoding these proteins are from a common / ancient gene family, further supporting the notion that TPCs present an intermediate between the 6-TM structured channels such as TRPs and the 4-repeat, 24-TM pore forming subunits of, for example, Nav and

Cav. Not only do TPCs exhibit a broad species distribution but they also appear to

have an extensive tissue distribution. Analysis of TPC2 tissue expression by Northern blot shows that TPC2 mRNA is present in most human tissues, including the cardiovascular system, with a particularly high level of expression in the liver and kidney (Fig. 4.1B), very similar to the tissue distribution of TPC1 in rat (Ishibashi et al., 2000). The broad range of tissue expression observed with TPC1 and TPC2 in mammals suggests a potentially important role for TPCs in cell function within the animal kingdom.

Fig. 4.3. Comparison of TPCs from multiple species

Comparison of the phylogenetic relationship between known TPC sequences plotted and aligned by my collaborator Dr. Mike Zhu using ClusterW (http://align.genome.jp) and plotted using a Neighbour-Joining algorithm. Representative sequences for the third and fourth TM domains of the pore-forming subunits of family members of CaV and NaV channels are also

included for comparison. GenBank accession numbers are shown in parentheses. * Indicates that corrections were made to these sequences based on multi-sequence alignments and splice donor and acceptor sites in genomic sequences. ** Indicates that sequence was assembled from genomic sequences. † Indicates that this sequence was previously designated as Ci- TPC1 (Okamura et al., 2005).