CALCIUM SIGNALING, ION CHANNELS AND MORE

Abstract

The DT40 B-lymphocyte cell line is a chicken bursal lymphocyte tumor cell line which grows rapidly, expresses a variety of types of constitutive and signal dependent ion transport systems., and supports the efficient use of stable and conditional genetic manipulations. Below, we review the use of DT40 cells in dissecting molecular mechanisms involved in Ca²⁺, Mg²⁺, and Zn²⁺ transport physiology. These studies highlight the flexibility and advantages the DT40 environment offers to investigators interested in the study of basic vertebrate ion transport physiology.

1.2 Genetic analysis of divalent cation physiology in DT40 cells

Divalent cations are ubiquitously involved in the cell physiology of living organisms, and in vertebrates play a multitude of roles ranging from promoting protein folding and maintenance of protein tertiary structures, cofactor to cellular nucleotides, participation in catalysis, and signal transduction. Because of their importance to such diverse processes, the regulatory mechanisms of the major divalent cations Ca²⁺, Mg²⁺, and Zn²⁺ have been the subject of investigation for many years. While the use of DT40 cells to study cation channel physiology has been slow to catch on, the past several years have seen an acceleration of their use in investigation of vertebrate ion homeostasis (fig 1 and Tab 1). Below, the application of genetically manipulated DT40 cells in the study of the physiology of the three major cellular divalent cations is reviewed, with an emphasis on how data from DT40 cells has made unique contributions to our understanding of the cellular roles of specific proteins in divalent cation physiology.

Figure #15-1.

The known DT40 channelome: The genes encoding channels and transporters in bold italic have been deleted in the DT40 system, and the phenotype published.

Table #15-1.

The DT40 “Channelome”: Overview of the expression pattern of ion channels and transporters in the DT40 cell line

Name	Major features	DT40 Expression, ref	DT40 KO phenotype, ref
CATIONIC CHANNELS
TRPC1	Regulated by PLC, store-depletion? Non-selective, Ca2+-permeable	YES	Both Ca2+ release-activated Ca2+ currents and IP3-mediated Ca2+ release from endoplasmic reticulum (ER) are reduced, resulting in decreased B cell antigen receptor-mediated Ca2+ oscillations and NFAT activation (Mori et al, 2002).
		MORI ET AL (2002)
TRPC2	Regulated by PLC, DAG, store-depletion? Non-selective, Ca2+-permeable	No representation in the DT40/Bursa EST database Note:dkfz426_12a16r 1.blsp and dkfz426_14n8r1.blge do not appear to be real matches to TRPC2 sequences	N/A
TRPC3-TRPC6	Non-selective, Ca2+-permeable	No representation in the DT40/Bursa EST database	N/A
TRPC7	Regulated by PLC, DAG, store-depletion? Non-selective, Ca2+-permeable	YES	No diminution in whole-cell ICRAC current, increased size of the Ca2+-stores. Non store-operated cation entry in response to the activation of the BCR, PAR2, intracellular dialysis with GTPγS, or application of DAG is absent (Lievremont et al, 2005)
		Lievremont et al (2005)
TRPV channels		No representation in the DT40/Bursa EST database	N/A
TRPM1	?	Yes (Kurosaki, Scharenberg, Perraud, unpublished)	N/A
TRPM2	C-terminal Nudix ADP-ribose hydrolase Gated by: ADP-ribose, oxidants Non-selective, Ca2+-permeable	No (Scharenberg, Perraud, unpublished) Note: riken1_9j1r1 corresponds to the chicken TRPM8 homologue, and not to TRPM2 as suggested.	N/A
TRPM3	activated by sphingolipids? Ca2+-permeable	No representation in the DT40/Bursa EST database	N/A
TRPM4	Gated by Ca2+, voltage-sensitive Monovalent specific	No (Scharenberg, Perraud, unpublished)	N/A
TRPM5	Gated by Ca2+, voltage-sensitive Monovalent specific	No representation in the DT40/Bursa EST database	N/A
TRPM6	C-terminal α-kinase? Mg2+	No (Scharenberg, Perraud, unpublished)	N/A
TRPM7	C-terminal α-kinase inhibited by intracellular Mg2+/Mg.nucleotides Divalent cation selective (Mg2+, Ca2+…)	YES	Homozygous deletion lethal unless medium supplemented with mM Mg2+. In the absence of supplementary Mg2+, total cellular Mg2+ drops, and the cells go into growth arrest and ultimately die (Schmitz et al, 2003).
		Nadler et al (2001), Schmitz et al (2003)
TRPM8	Cold, menthol, icilin Non-selective, Ca2+-permeable	No (Scharenberg, Perraud, unpublished) Note: riken1_9j1r1 corresponds to TRPM8, but we have not been able to confirm DT40 expression of cTRPM8 by RT-PCR	N/A
TPC1 and TPC2	Two-pore channels 1and 2 Ca2+/Na+ permeable	Yes (Ping Liu and A.M. Scharenberg, unpublished)	N/A
IP3-receptors type I, II, III	Intracellular (ER Ca2+-store membrane) IP3-gated Ca2+ channels	YES	DT40s in which a single type of IP3R has been deleted still mobilize Ca2+ in response to BCR stimulation, whereas this Ca2+ mobilization is abrogated in B cells lacking all three types of IP3R (Sugawara et al, 1997). IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients (Miyakawa et al, 1999).
		Sugawara et al (1997)
Ryanodine receptors, type 1 and 3	Intracellular (ER Ca2+-store membrane) Ca2+-channels	Yes (no Type 2) Kiselyov et al (2001)	N/A
KCNK1 (TWIK-1)	Two-pore domain K+ channel	Represented in the DT40/Bursa EST database: riken1_6b3r1; riken1_6b3	N/A
Kv channels and other K+-channels		No convincing matches could be identified in the DT40/Bursa EST database
SCN8A	Na+ channel, voltage gated, type VIII, alpha	Represented in the DT40/Bursa EST database: dkfz426_25g18r1	N/A
ZnT5, ZnT6, ZnT7	Zinc-transporter, mainly in the Golgi and vesicular subcompartment	Suzuki et al (2005)	ZnT5−/−/ZnT6−/−/Znt7−/− look healthy and total cellular Zn2+ like in WT, confirming that these transporters supply Zn2+ into the lumens of the secretory compartments. Activity og Golgi enzymes requiring Zn2+ severely reduced. Znt5 and ZnT6 associate and are in the same pathway, Znt7 works alone.
SLC41A2	Mg2+ transporter, possible transporter of other divalent cations	Yes, J. Sahni and A. M. Scharenberg, unpublished	N/A
ANIONIC CHANNELS
ClC-3	Voltage-gated chloride channel	Represented in the DT40/Bursa EST database: riken1_4d21r1	N/A
ClC-5	Voltage-gated chloride channel Cl-/H+ exchangers	Represented in the DT40/Bursa EST database: dkfz426_4i15r1	N/A
ClC-7	endosomal/lysosomal chloride channel	riken1_7i18r1	N/A
CFTR	ABC (ATP-binding cassette)-transporter; cAMP dependent chloride channel	No representation in the DT40/Bursa EST database dkfz426_8l16r1 and riken1_17b24r1.blsp (corresponds tp the multidrug transporter ABCC4) do not appear to be a real match to CFTR sequences.	N/A
CLIC2	Intracellular chloride channel? Inhibits RyR, might limit Ca2+ release from internal stores	Represented in the DT40/Bursa EST database: riken1_10k5r1	N/A
VDAC 2	Predominantly expressed in the outer mitochondrial membrane; almost freely permeable to low molecular-weight molecules, slight preference for anions over cations	Represented in the DT40/Bursa EST database: dkfz426_15a24r1	N/A
VDAC3	Same as above	Represented in the DT40/Bursa EST database: dkfz426_1j21r1	N/A

Note: Only one EST clone ID is indicated per gene, although there are for all the listed genes multiple entries. Overlapping clones can be easily identified in the database.

Kiselyov K, Shin DM, Shcheynikov N, Kurosaki T, Muallem S (2001): Regulation of Ca²⁺-release-activated Ca²⁺ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells. Biochem J; 360(Pt 1):17–22.

Lievremont J-P, Numaga T, Vazquez G, Lemonnier L, Hara Y, Mori E, Trebak M, Moss SE, Bird GS, Mori Y, Putney JW Jr. (2005): The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels. J. Biol. Chem. 280: 35346–35351.

Miyakawa T, Maeda A, Yamazawa T, Hirose K, Kurosaki T, Iino M (1999): Encoding of Ca²⁺ signals by differential expression of IP3 receptor subtypes. EMBO J; 18(5):1303-8.

Mori Y, Wakamori M, Miyakawa T, Hermosura M, Hara Y, Nishida M, Hirose K, Mizushima A, Kurosaki M, Mori E, Gotoh K, Okada T, Fleig A, Penner R, Iino M, Kurosaki T (2002): Transient receptor potential 1 regulates capacitative Ca²⁺ entry and Ca²⁺ release from endoplasmic reticulum in B lymphocytes. J Exp Med; 195(6):673-81.

Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A (2001): LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature; 411(6837):590-5.

Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, Scharenberg AM (2003): Regulation of vertebrate cellular Mg²⁺ homeostasis by TRPM7.Cell;114(2):191–200.

Sugawara H, Kurosaki M, Takata M, Kurosaki T (1997): Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J; 16(11):3078-88.

Suzuki T, Ishihara K, Migaki H, Ishihara K, Nagao M, Yamaguchi-Iwai Y, Kambe T (2005): Two different zinc transport complexes of cation diffusion facilitator proteins localized in the secretory pathway operate to activate alkaline phosphatases in vertebrate cells. J Biol Chem; 280(35):30956-62.

1.2.1 Issues in unraveling regulation of ion homeostasis

Not surprisingly, regulation of divalent cation uptake and intracellular homeostasis is complex, particularly in higher organisms such as vertebrates, and involves a wide range of channels, transporters, and pumps specific for each type of cation. The complexity of higher organisms coupled with the fundamental roles which divalent cations play in many cellular processes has rendered the study of divalent cation transport (in the context of a living organism) using modern genetic techniques problematic due to the existence of multiple channel/transporter homologues, and the capacity of related channels to compensate through up or down regulation of their function. For example, animal models in which specific channels or transporters have been deleted have been useful in understanding the contribution of a protein to the development of the organism or to pathophysiologic processes manifesting at the whole organism level, but have been less useful in providing insights into molecular mechanisms which regulate particular channels/transporters, or to intracellular biochemical processes directly influenced by their function. These problems are further exacerbated by the costs of animal care and effort/time required to execute complex genetic manipulations at the whole animal level.

Because of the issues inherent in the application of genetic techniques at the whole animal level, a great deal of our present understanding of divalent cation channel physiology is derived from the use of pharmacologic inhibitors or inferred from the behavior of ion channels or transporters analyzed after heterologous expression in whole cell patch clamp experiments. While these techniques are indispensable for generating biophysical data on the function of ion channels and transporters, methods which allow the correlation of pharmacological effects and in-vitro data with the responses of intact cells are important for understanding how particular molecular functions of a channels and transporting proteins fit within the multitude of processes ongoing in living cells. It is here that the DT40 system stands out, as it offers physiologists and biophysicists a unique, inexpensive, and relatively simple system in which to combine physiological studies with the power of molecular genetics:

1. DT40 cells are derived from a vertebrate organism, and therefore findings in this system are relevant to a wide variety of higher organisms, including humans.

2. DT40 cells provide a reasonable model of the behavior of a continuously growing hematopoietic cell.

3. DT40 cells are large enough to be suitable for various forms of imaging, and to allow the application of patch clamp and other electrophysiologic methods without the need for isolation/dissection.

4. DT40 cells support the facile use of stable and conditional genetic modifications for manipulation of genes, either to produce conditional knockouts or for the purpose of more subtle manipulations to confer inhibitor activity to desired target ion channels.

1.2.1 Ca²⁺ physiology in DT40 cells

Tomohiro Kurosaki's group's investigation of the biochemical mechanisms activated by engagement of the B-cell antigen receptor was the first application of DT40 cell genetics to the study of signal transduction. Dr. Kurosaki's body of work using the DT40 system is a noteworthy example of what can be accomplished using the DT40 system to systematically apply physiology, biochemistry and genetics to unravel a biological puzzle. As an early elevation of cytosolic Ca²⁺ is an important component of the signal produced by BCR engagement, this work is also an important contribution to the present knowledge of vertebrate divalent cation physiology.

Dr. Kurosaki's group's first major contribution in this area was the demonstration that IP3 receptors are absolutely required for both BCR-induced calcium release and calcium entry, but are dispensable for the activation of store operated calcium entry. This observation firmly established that BCR-mediated IP3 production has a crucial role in the calcium entry required for generation of a sustained calcium signal (Sugawara et al., 1997). The technical achievement of producing this cell line was also significant, as it required deletion of both alleles of each of three IP3 receptor genes using six different resistance markers to generate IP3R-triple knockout cells - the first time that any cell type had been so extensively genetically manipulated. These cells have now been utilized by laboratory's around the world to study the role which IP3-receptors have in a variety of cell physiological or receptor-mediated signaling processes, such as the IP3 receptor-independent regulation of TRPC channels and the role of ryanodine receptors in activation of store operated CRAC channels (Broad et al., 2001; Bultynck et al., 2004; Cui et al., 2004; Guillemette et al., 2005; Kiselyov et al., 2001; Laude et al., 2005; Ma et al., 2001; Ma et al., 2002; Morita et al., 2004; Prakriya and Lewis, 2001; Vazquez et al., 2001; Vazquez et al., 2002; Vazquez et al., 2003; Venkatachalam et al., 2001; Wedel et al., 2003; Yogo et al., 2004).

A second important area of study undertaken by Dr. Kurosaki's laboratory in conjunction with the laboratory of Dr. Yasuo Mori has been a genetic analysis of the role of TRPC ion channels in BCR-activated and store-operated calcium signals in vertebrate cells. Prior to work in the DT40 system, multiple studies on TRPC channel gating mechanisms had provided a diverse set of conflicting data on TRPC channel function. However, by demonstrating that knockout of the TRPC1 gene in DT40 cells left store operated calcium entry intact, but produced alterations in both BCR-induced calcium release and calcium entry, this work provided one of the first pieces of evidence placing TRP channel function at the level of IP3 production, as opposed to direct participation in store operated calcium entry (Mori et al., 2002). Subsequent analysis of a TRPC7-knockout DT40 line by the Mori and Putney groups has also implicated TRPC7 channels in receptor-operated, but not store operated cation entry (Lievremont et al., 2005). These data have provided important supportive evidence that TRPC1 and TRPC7 channels probably do not underly CRAC channel activity, although subtle alterations in the measurement of CRAC currents in TRPC1 knockout cells leave open the possibility of some linkage between TRPC proteins and CRAC channels.

A final area in which DT40 cell genetics have made an important contribution to understanding the physiology of calcium signaling is in defining the role of TRPM7, a ubiquitous ion channel which underlies MagNuM/MIC currents present in many types of cells (Nadler et al., 2001). As TRPM7 is a member of the TRP superfamily, it had been assumed that it was primarily permeating Na⁺ and Ca²⁺, A collaboration between our group and Dr. Kurosaki's group led the creation of a conditional knockout of TRPM7. Analysis of this cell line demonstrated that TRPM7 was required for cell growth, and to a later observation that TRPM7-deficient cells could be grown if sufficient extracellular Mg²⁺ were provided (discussed in more detail below (Nadler et al., 2001; Schmitz et al., 2004)). The capacity to grow TRPM7-deficient cells has allowed us to study their Ca²⁺ signaling physiology, and we have observed no apparent differences between these cells and WT DT40 cells, providing evidence against a major role for TRPM7 in receptor-mediated or store-operated calcium signaling (C. Schmitz, A-L Perraud, and A.M. Scharenberg, unpublished data).

1.2.2 Mg²⁺ physiology in DT40 cells

As mentioned above, the generation of a knockout of TRPM7 in DT40 cells led to the observation that TRPM7-deficient DT40 cells are not able to grow in regular media, providing the first demonstration that an ion channel has a crucial role in the regulation of cell growth. Subsequently, a homologue of TRPM7, TRPM6, was shown to have a role in the regulation of organismal Mg²⁺ homeostasis, and this observation led to our own exploration of whether TRPM7 might play an analogous role in Mg²⁺ homeostasis at the cellular level (Schmitz et al., 2004). Our studies have shown that TRPM7-deficient DT40 cells can be grown indefinitely if provided with supplemental Mg²⁺ in their media. We have built on this observation by utilizing the capacity of DT40 cells to allow conditional expression of mutant forms of TRPM7 using a Tet-suppressor based conditional expression approach (Schmitz et al., 2004). These studies have shown that the capacity of TRPM7 channels to complement cell growth correlates with their net capacity to transport Mg²⁺ across the plasma membrane (i.e. the number of channels and/or their sensitivity to suppression by free Mg²⁺ determines their capacity to support cell prolifertion), but is independent of their intrinsic protein kinase activity. Overall these studies support a key role for TRPM7 in the regulation of DT40 cell Mg²⁺ homeostasis. We have subsequently utilized our TRPM7-deficient cells to show that SLC41 proteins, which have distant homology to a class of prokaryotic Mg²⁺ transporters, are able to also complement the growth defect of TRPM7-deficient cells (J. Sahni and A. M. Scharenberg, in preparation). This observation further supports the involvement of TRPM7 in Mg²⁺ uptake, and also provides the first evidence that SLC41 proteins have a role in vertebrate cellular Mg²⁺ uptake.

My laboratory is presently utilizing the DT40 system to further analyze mechanisms which regulate Mg²⁺ homeostasis by attempting to create both stable and conditional knockouts of SLC41 proteins. Furthermore, we are exploiting another advantage of DT40 cells, which is the capacity to control their extracellular ionic environment to better understand how transport of a particular ion is regulated. As an example, we have determined that DT40 cells are able to grow at a normal rate when extracellular Mg²⁺ is lowered to 100 µM, or when 95 mM KCl is substituted for the normal 95 mM NaCl in their extracellular media (J. Sahni and A. M. Scharenberg, in preparation). The latter substitution largely collapses the membrane potential, enhancing DT40 cells' dependence on chemical gradients to drive uptake of individual nutrients and ions. However, under the collapsed Vmem conditions, DT40 cells grow at a normal rate if Mg²⁺ is at a physiologic level of 0.5 mM, but their rate of growth slows in proportion to the extracellular Mg²⁺ concentration if the extracellular Mg²⁺ concentration is dropped below 0.4-.5 mM free Mg²⁺, in the range of what free [Mg²⁺] is thought to be inside the cell. This observation strongly suggests that the majority of DT40 Mg²⁺ uptake occurs via a Vmem dependent mechanism, consistent with a major contribution from an ion channel transport mechanism such as TRPM7.

1.2.3 Zn²⁺ physiology in DT40 cells

Most recently, Suzuki, Kambe and colleagues have been exploring the use of DT40 cells in analysis of Zn²⁺ transport and homeostasis in vertebrate cells (Suzuki et al., 2005a; Suzuki et al., 2005b). They have utilized DT40 genetics to evaluate the role of the ZnT5, ZnT6, and ZnT7 zinc transporters by producing ZnT5, ZnT6, and ZnT7 single, double, and triple knockout cell lines (Suzuki et al., 2005a; Suzuki et al., 2005b). Using these cell lines, they have performed analyses of vertebrate Zn²⁺ regulatory mechanisms which demonstrate that ZnT5 and ZnT7 transporters have overlapping functions in loading zinc into the Golgi lumen for assembly with enzymes such as alklaline phosphatase apoenzymes, which must bind Zn²⁺ prior to maturing into their active forms. They have also utilized re-expression of ZnT5, ZnT6, and ZnT7 proteins to show that ZnT5 and ZnT6 work in a similar pathway and hetero-oligomerize, and that each protein may complement the function of the other, while ZnT7 works in a different pathway as a homo-oligomer. In combination, these data provide clear evidence on the function of ZnT5, ZnT6, and ZnT7 in vertebrate Zn²⁺ physiology at the cellular level. They also illustrate the utility which DT40 cells have as a model for the study of the individual contributions which various Zn²⁺ transporters make to maintaining Zn²⁺ homeostasis in vertebrate cells.

1.3 Summary

Involvement of ion transport proteins in diverse aspects of organism physiology in many cases renders the analysis of their regulation and role in cell physiology through the creation of organism level-knockouts either prohibitively expensive or non-feasible due to issues of profoundly altered organism development or complete non-viability. However, in the DT40 system, genetic manipulations have been achieved for up to three distinct homologues (six different alleles) of a given protein family, and have allowed a number of detailed studies to be performed on cells with well defined defects in specific cation transport pathways. In the future, the use of floxed selection markers that can be recycled by excision through the Cre-recombinase should allow the deletion of virtually any desired number of genes of interest in the same DT40 cell line. Furthermore, the stable phenotype of genetically manipulated DT40 cells coupled with their capacity to be transfected with mutant forms of deficient proteins allows "structure/function" analyses to be performed with a functional readout - an important issue for proteins such as transporters and channels where in-vitro functional assays may not be available. Overall, the DT40 system offers a well defined vertebrate system where modern genetic manipulations can be combined with electrophysiological and cell biological methods to gain novel insights into the cell physiology of ion transport.