Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca(2+) is required for both structural and biophysical roles. In addition, changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) orchestrate responses to developmental and environmental signals. In many instances, [Ca(2+)](cyt) is increased by Ca(2+) influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca(2+)-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca(2+)-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca(2+)-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca(2+) channels, the genetic counterparts of specific Ca(2+)-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca(2+) channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca(2+) channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La(3+) and nifedipine, and their increased activity as [Ca(2+)](cyt) is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca(2+)-permeable) K(+) channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses.

The putative two-pore Ca(2+) channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co-localizes with the K(+)-selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca(2+)-activated slow vacuolar (SV) currents, which were increased in AtTPC1-over-expressing Arabidopsis compared to the wild-type. A Ca(2+)-insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1-2 knockout plants. The kinetics of ABA- and CO(2)-induced stomatal closure were similar in wild-type and tpc1-2 knockout plants, excluding a role of SV channels in guard-cell signalling in response to these physiological stimuli. ABA-, K(+)-, and Ca(2+)-dependent root growth phenotypes were not changed in tpc1-2 compared to wild-type plants. Given the permeability of SV channels to mono- and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca(2+) entry into the cytosol. Ca(2+) responses as measured in aequorin-expressing wild-type, tpc1-2 knockout and TPC1-over-expressing plants disprove a contribution of TPC1 to any of the stimulus-induced Ca(2+) signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca(2+) concentration and biotic factors (elf18, flg22). In good agreement, stimulus- and Ca(2+)-dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization-activated Ca(2+)-permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca(2+) homeostasis.     

Aluminum as a specific inhibitor of plant TPC1 Ca2+ channels

In plant cells, Al ion plays dual roles as an inducer and an inhibitor of Ca(2+) influx depending on the concentration. Here, the effects of Al on Ca(2+) signaling were assessed in tobacco BY-2 cells expressing aequorin and a putative plant Ca(2+) channel from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). In wild-type cells (expressing only aequorin), Al treatment induced the generation of superoxide, and Ca(2+) influx was secondarily induced by superoxide. Higher Al concentrations inhibited the Al-stimulated and superoxide-mediated Ca(2+) influx, indicating that Ca(2+) channels responsive to reactive oxygen species (ROS) are blocked by high concentration of Al. H(2)O(2)-induced Ca(2+) influx was also inhibited by Al. Thus, inhibitory action of Al against ROS-induced Ca(2+) influx was confirmed. Similarly, known Ca(2+) channel blockers such as ions of La and Gd inhibited the H(2)O(2)-induced Ca(2+) influx. While La also inhibited the hypoosmotically induced Ca(2+) influx, Al showed no inhibitory effect against the hypoosmotic Ca(2+) influx. The effects of Al and La on Ca(2+) influx were also tested in the cell line overexpressing AtTPC1 and the cell line AtTPC1-dependently cosuppressing the endogenous TPC1 equivalents. Notably, responsiveness to H(2)O(2) was lost in the cosuppression cell line, thus TPC1 channels are required for ROS-responsive Ca(2+) influx. Data also suggested that hypoosmotic shock induces TPC1-independent Ca(2+) influx and Al shows no inhibitory action against the TPC1-independent event. In addition, AtTPC1 overexpression resulted in a marked increase in Al-sensitive Ca(2+) influx, indicating that TPC1 channels participate in osmotic Ca(2+) influx only when overexpressed. We concluded that members of TPC1 channel family are the only ROS-responsive Ca(2+) channels and are the possible targets of Al-dependent inhibition.     

Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1

We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.     

High glucose alters apoptosis and proliferation in HEK293 cells by inhibition of cloned BK Ca channel

It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+β1 subunit of BK(Ca) channel, hSloα+β1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+β1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+β1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+β1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.     

Action of aluminum, novel TPC1-type channel inhibitor, against salicylate-induced and cold-shock-induced calcium influx in tobacco BY-2 cells

Previously, effect of Al ions on calcium signaling was assessed in tobacco cells expressing a Ca2+-monitoring luminescent protein, aequorin and a newly isolated putative plant Ca2+ channel protein from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). TPC1 channels were shown to be the only channel known to be sensitive to Al and they are responsive to reactive oxygen species and cryptogein, a fungal elicitor protein. Thus, involvement of TPC1 channels in calcium signaling leading to development of plant defense mechanism has been suggested. Then, the use of Al as a specific inhibitor of TPC1-type plant calcium channels has been proposed. Here, using transgenic tobacco BY-2 cells expressing aequorin, we report on the evidence in support of the involvement of Al-sensitive signaling pathway requiring TPC1-type channel-dependent Ca2+ influx in response to salicylic acid, a key plant defense-inducing agent, but not to an elicitor prepared from the cell wall of rice blast disease fungus Magnaporthe grisea. In addition, involvement of Al-sensitive Ca2+ channels in response to cold shock was also tested. The data suggested that the elicitor used here induces the Ca2+ influx via Al-insensitive path, while salicylic acid and cold-shock-stimulate the influx of Ca2+ via Al-sensitive mechanism.     

Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

The ascidian dihydropyridine-resistant calcium channel as the prototype of chordate L-type calcium channel

This review describes recent findings on voltage-gated Ca channel (Cav channel) cloned from ascidians, the most primitive chordates. Ascidian L-type like Cav channel has several unusual features: (1). it is closely related to the prototype of chordate L-type Cav channels by sequence alignment; (2). it is resistant to dihydropyridine due to single amino acid change in the pore region, and (3). maternally provided RNA putatively encodes a truncated protein which has remarkable suppressive effect on Cav channel expression during development. Ascidian Cav channel will provide a useful molecular clue in the future to understand Ca(2+)-regulated cell differentiation and physiology with the background of recently defined ascidian genome and molecular biological tools.     

Plant K+ channel alpha-subunits assemble indiscriminately.

In plants a large diversity of inwardly rectifying K+ channels (K(in) channels) has been observed between tissues and species. However, only three different types of voltage-dependent plant K+ uptake channel subfamilies have been cloned so far; they relate either to KAT1, AKT1, or AtKC1. To explore the mechanisms underlying the channel diversity, we investigated the assembly of plant inwardly rectifying alpha-subunits. cRNA encoding five different K+ channel alpha-subunits of the three subfamilies (KAT1, KST1, AKT1, SKT1, and AtKC1) which were isolated from different tissues, species, and plant families (Arabidopsis thaliana and Solanum tuberosum) was reciprocally co-injected into Xenopus oocytes. We identified plant K+ channels as multimers. Moreover, using K+ channel mutants expressing different sensitivities to voltage, Cs+, Ca2+, and H+, we could prove heteromers on the basis of their altered voltage and modulator susceptibility. We discovered that, in contrast to animal K+ channel alpha-subunits, functional aggregates of plant K(in) channel alpha-subunits assembled indiscriminately. Interestingly, AKT-type channels from A. thaliana and S. tuberosum, which as homomers were electrically silent in oocytes after co-expression, mediated K+ currents. Our findings suggest that K+ channel diversity in plants results from nonselective heteromerization of different alpha-subunits, and thus depends on the spatial segregation of individual alpha-subunit pools and the degree of temporal overlap and kinetics of expression.     

The Ca2+ Status of the Endoplasmic Reticulum Is Altered by Induction of Calreticulin Expression in Transgenic Plants

To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.     

The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel

The epithelial Ca(2+) channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca(2+) (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca(2+) permeation of ECaC and modulation by extracellular Mg(2+). Mutation of the aspartate residue, D542A, abolishes Ca(2+) permeation and Ca(2+)-dependent current decay as well as block by extracellular Mg(2+), whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca(2+) permeability and Ca(2+)-dependent current decay. Block of monovalent currents through ECaC by Mg(2+) was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e. Glu(535) and Asp(550), had only minor effects on Ca(2+) permeation properties.     

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.