Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

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Abstract

P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca2+, Na+, K+ and H+), have been reported. They include reticulum and plasma-membrane Ca2+-ATPases, Na+/K+-ATPase and H+/K+-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg2+ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na+/K+-ATPase α1-isoform, H+/K+-ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H+/K+-ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

Introduction

The P-type ATPases constitute a family of integral membrane proteins, which actively transport cations or amino-phospholipids across membrane, coupling this process with ATP-hydrolysis [1], [2]. They are also denominated E1–E2 ATPases because they follow a characteristic reaction cycle between two different conformational states: E1 and E2 [2]. During this catalytic cycle, γ-phosphate group from ATP is transferred to a specific aspartyl-residue of the enzyme, generating a phospho-enzyme intermediary, which is characteristic of all P-type ATPases [1], [2]. This catalysis is Mg2+-dependent and inhibited by vanadate [1], [2].

At least, ten different subtypes of P-type ATPases have been described [2], [3]. The subtypes II-A, II-B and II-C are the only, described in mammalia, that transport low-atomic-weight cations [2], [3]. Subtype II-A is integrated by Ca2+-ATPases from sarco/endo-plasmatic reticulum (SERCA) and Golgi apparatus (PMR). Three isoforms of SERCA (SERCA1-3) and two isoforms of PMR (PMR1-2) have been identified. Subtype II-B includes Ca2+-ATPases from plasma membrane, existing four PMCA isoforms (PMCA1-4). Subtype II-C is mainly integrated by Cation/K+-ATPases, which include four isoforms of the Na+/K+-ATPase (ATP1A1-4) and two isoforms of H+/K+-ATPase, the gastric (ATP4A) and non-gastric (ATP12A or ATP1AL1) isoforms [2], [3]. Until now, only SERCA3, PMCA1, PMCA4, ATP1A1 and ATP12A have been identified in intestinal epithelia [4], [5], [6]. However, the genetic identity of certain P-type ATPase activities, described in intestinal epithelial cells [7], [8], has not been elucidated.

The catalytic subunit of P-type ATPases (α-subunit) has 6–10 α-helical transmembrane segments (M1–M10) alternated with cytosolic and extra-cytosolic loops [1], [2]. The cytosolic B-loop, between M2 and M3, contains the phosphatase domain (A-domain). The cytosolic C-loop, located between M4 and M5, includes the kinase and the nucleotide-binding domains (P- and N-domains, respectively) [2]. Additionally, P-type ATPases have nine highly-preserved structural motifs, denoted with letters from A to I, which are arranged from N- to C-terminal as follows: “A” (PGDX10PAD), “B” (TGES), “C” (GX9G), “D” (P[E/V/I/C]GL), “E” (ICSDKTGTLT), “F” (KGAP), “G” (DPPRX6[I/V]X6GX6TGDX4A), “H” (TGDGVNDSPALKKAD), and “I” (A[K/R]XAAD) [1], [2], [3]. The C-loop has five of them, including the two most preserved: E (phosphorylation-motif) and H (Mg2+-ATP binding motif) [1], [2], [3]. The E-motif includes the essential Asp residue phosphorylated during the catalytic cycle [1], [2].

P-type ATPases are quasi-ubiquitously distributed into phylogenetic scale [1], [2], [3]. They are essential for several physiological processes, have been involved in some pathologies and are important therapeutic-targets [2], [9], [10]. In intestinal epithelia, P-type ATPases play essential roles in absorptive and secretory processes [6], [7], [8], [11], [12], [13]. Moreover, enterocytes and colonocytes show some functional differences which seem to be partially due to the differential expression of these enzymes [8], [12]. The recognition of cell-specific expression pattern (or the identification of new P-type ATPases) is essential for the understanding of mechanisms involved in intestinal function. Thus, their detection by specific and sensitive techniques becomes relevant.

Degenerate-PCR, based in conserved amino-acid sequences, has been successfully employed to clone several genes [14], [15]. This technique requires two target amino-acid sequences, of at least 6-amino-acid long, which must be highly preserved and adequately located at the protein primary structure [14]. P-type ATPases only have two motifs that fulfill these requirements: the E- and H-motifs.

In this paper, expression of P-type ATPases from subtypes II is determined in enterocytes and colonocytes by a degenerate-PCR variant denominated Multiplex-Nested-PCR (MN-PCR). It combines successive RT-PCR and Nested-PCR to enhance specificity and sensibility. Both PCRs employed degenerate primers, which recognize the cDNA-segments that encode the E- and H-motifs. Additionally, the size of the PCR-products was specific for each P-type ATPase.

Section snippets

Materials and methods

Present work was approved by IVIC Bioethical Committee and carried out in accordance with EC Directive 86/609/EEC for animal experiments.

Materials. Agarose, Trizol Reagent, Thermoscript RT-PCR System, HiFi Platinum Taq DNA Polymerase, TOPO TA Cloning Kit for Sequencing, DNA weight markers, antibiotics, culture mediums and custom primers were from Invitrogen. Thermocyclers, electrophoretic reagents and devices were from Bio-Rad and MJ Research. Real-time master mix was from Fynnzymes. Wizard

Results

RT-PCR and Nested-PCR were carried out to identify the expression of P-type ATPase mRNAs, from subtypes II, in intestinal epithelial cells.

Fig. 2A shows the 4% PAGE of RT-PCR and Nested-PCR products from enterocytes (E) and colonocytes (C). Four DNA-bands were detected by RT-PCR in enterocytes (E1’–E4’) and five DNA-bands in colonocytes (C1’–C5’). Likewise, Nested-PCR generated four DNA-bands in enterocytes (E1–E4) and five DNA-bands in colonocytes (C1–C5). In general, Nested-PCR bands showed a

Discussion

Different P-type ATPase activities, with diverse ion-specificity, affinity, regulation and cell distribution, have been reported in intestinal epithelia [4], [5], [6], [7], [8], [11], [12], [13]. However, some of them have not been associated with a specific gene [7], [8]. Until now, 15 genes of P-type ATPases that transport low-atomic-weight cations (Ca2+, Na+, K+ and H+) have been reported in mammalia [2], [3]. These enzymes present a tissue-specific distribution with physiological

Conclusions

Epithelial cells of small intestine and distal colon have functional differences that are partially due to the differential expression of P-type ATPases. Enterocytes, which actively absorb calcium, preferentially expressed PMCA4. In contrast, ATP12A was differentially expressed in colonocytes that actively absorb potassium. Several P-type ATPases are shared by both cellular types, including a putative new splice-variant of atp1a1 gene.

Acknowledgments

We thank Dr. Howard Takiff, MSc., María Sulbarán, Dr. Flor Pujol and Lic. Carmen Loureiro for their cooperation. This work was supported by Grants from FONACIT (Project Numbers: F-2005000222 and S1-2000000552) and MISIÓN CIENCIA (Project Number: 20070001585).

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