CASTOR is a multi-pass membrane cation channel of the CASTOR/POLLUX/DMI1 family that resides in the nuclear envelope of Lotus japonicus root cells. Each subunit has a pore-forming transmembrane region and a large soluble C-terminal region made of two tandem RCK (regulator of conductance for K+) domains; four subunits assemble a Ca2+-binding RCK gating ring, and CASTOR and its paralog POLLUX form separate homo-oligomeric channels. Reconstituted full-length CASTOR is a high-conductance, weakly selective cation channel permeable to K+, Na+ and Ca2+, blocked by Mg2+ at negative voltages; a truncated pore construct studied by patch clamp was strongly Ca2+-selective and activated by cytosolic/nucleosolic Ca2+. CASTOR, together with POLLUX and the nuclear-envelope Ca2+ channels CNGC15, is required to generate the perinuclear/nuclear Ca2+ oscillations ("Ca2+ spiking") that are triggered by rhizobial Nod factors and arbuscular mycorrhizal fungal signals and decoded by CCaMK. Its widely held role is as a K+-permeable counter-ion channel that balances charge during Ca2+ release from the nuclear envelope lumen, although direct Ca2+ conduction has not been excluded. castor mutants retain the initial Nod factor-induced root-hair Ca2+ influx but lack nuclear Ca2+ spiking, fail to allow rhizobial and fungal entry into root cells, and are defective in both root nodule symbiosis and arbuscular mycorrhiza.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006811 monoatomic ion transport | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the CASTOR/POLLUX/SYM8-like family (IPR044849). CASTOR is an experimentally demonstrated ion channel: reconstituted CASTOR conducts monovalent cations and Ca2+ in planar lipid bilayers (PMID:19106374), and a pore construct conducts Na+ and Ca2+ in patch clamp (PMID:31420535). Reason: The term is broad but correct; CASTOR transports monoatomic cations across the nuclear envelope. It is consistent with, and less specific than, the cation channel activity proposed below. Because the dominant physiological permeant ion (K+ versus Ca2+) is still debated, this general process term is a safe family-level statement and need not be refined. Supporting Evidence: PMID:19106374 CASTOR reconstituted in planar lipid bilayers exhibited ion channel activity, and the channel characteristics were altered in a symbiosis-defective mutant carrying an amino acid replacement close to the selectivity filter. PMID:31420535 Figure 1a shows the whole-cell current in the presence of symmetrical 150 mM Na+, illustrating the cationic (Na+) currents conducted by LjCASTOR-2TM. |
| GO:0006813 potassium ion transport | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the RCK_N domain (IPR003148). The RCK domain alone does not determine selectivity (it is a gating/ligand-binding module shared by K+ and other channels), but for CASTOR K+ permeation is experimentally supported: reconstituted full-length CASTOR shows a weak preference for K+ over Na+ and Ca2+ (PMID:19106374), and K+ flux through CASTOR/POLLUX is the basis of the widely used counter-ion model of symbiotic nuclear Ca2+ spiking (PMID:27230377). Reason: Although reached by a weak domain-based route, the conclusion is supported by direct bilayer measurements on full-length CASTOR. The authors argue that K+, the most abundant cellular cation, is likely the main ion carried in vivo, and the later CNGC15 work refers to the DMI1/CASTOR/POLLUX channel as the potassium-permeable channel required for oscillations. Kim et al. (PMID:31420535) report that a truncated, plasma-membrane-retargeted CASTOR construct is highly Ca2+-selective, which challenges the K+ assignment. That construct is not the native full-length channel in the nuclear envelope, and the 2008 data still show K+ permeability. K+ transport is therefore retained, with the dominant physiological ion flagged as unresolved in suggested_questions. Supporting Evidence: PMID:19106374 Considering that potassium is the cation with the highest concentration within the cell, together with the permeability of CASTOR and POLLUX for potassium, we conclude that potassium is likely to be the major ion going through these cation channels in vivo. PMID:27230377 A potassium-permeable channel is known to be required for symbiotic Ca(2+) oscillations, but the calcium channels themselves have been unknown until now. PMID:31420535 Here we show that CASTOR from Lotus japonicus is a highly selective Ca2+ channel whose activation requires cytosolic/nucleosolic Ca2+, contrary to the previous suggestion of it being a K+ channel. |
| GO:0031965 nuclear membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Nucleus membrane). Immunogold electron microscopy with a CASTOR-specific antibody placed endogenous CASTOR at the nuclear envelope of wild-type Lotus root cells, with no labeling in the castor-12 premature-stop mutant. Earlier plastid localization of overexpressed fusions (PMID:15616514) is considered an overexpression artifact. Reason: Directly supported by localization of the native protein. The method could not resolve inner versus outer nuclear membrane, so the general nuclear membrane term is the right level of specificity. Plastid localization should not be annotated. Supporting Evidence: PMID:19106374 Immunogold labeling localized the endogenous CASTOR protein to the nuclear envelope of Lotus root cells. PMID:19106374 Therefore, we could not determine whether CASTOR resides in the inner or outer membrane of the nuclear envelope or in both. Only nonspecific background labeling was observed in plastids in both the wild type and castor-12 |
| GO:0005261 monoatomic cation channel activity | IDA PMID:19106374 Lotus japonicus CASTOR and POLLUX are ion channels essential... | NEW | Summary: Purified CASTOR reconstituted into planar lipid bilayers forms a high-conductance (about 260 pS) cation-selective channel. The channel is weakly selective among K+, Na+ and Ca2+, is blocked by Mg2+ at negative voltage, and its properties are altered by the symbiosis-defective castor-2 pore-region substitution. Patch-clamp recordings of a CASTOR pore construct confirm cation channel activity, and point mutations that abolish channel activity also abolish nodulation (PMID:31420535). Reason: The core molecular activity of CASTOR has no molecular-function annotation in GOA. Cation channel activity is directly demonstrated by two independent laboratories. The general cation term is used because the two studies disagree on selectivity (K+ preference in bilayers of full-length protein, PK/PCa about 1.4; strong Ca2+ selectivity of a truncated construct). Neither potassium channel activity nor calcium channel activity can yet be asserted for the native channel without taking sides in an open debate. Supporting Evidence: PMID:19106374 Permeability ratio determination and competition experiments reveled a weak preference of CASTOR for cations such as potassium over anions. PMID:19106374 In the absence of magnesium, the current–voltage relationship of CASTOR was linear over this voltage range with a conductance of 260 pS PMID:31420535 As expected, E493A mutant has much lower single-channel activity than wild-type channel, whereas no channel activity was observed for E493Q mutant, indicating that the loss of nodulation and Ca2+ spiking in Ca(II) site mutations can be attributed to the loss of channel function. |
| GO:0009877 nodulation | IMP PMID:31420535 Ca(2+)-regulated Ca(2+) channels with an RCK gating ring con... | NEW | Summary: castor mutants fail to allow rhizobial entry into root cells and do not nodulate. Wild-type CASTOR restores nodulation of a CASTOR-null line, whereas channel-dead Ca2+-site mutants (D442A, E493A, E493Q) do not. CASTOR channel activity generates the nuclear Ca2+ spikes that drive the symbiotic transcriptional program. Reason: Participation test: CASTOR does part of the work of the process. Its channel activity is a mechanistic step in generating the nuclear Ca2+ oscillations of the common symbiosis signaling pathway, and nodulation depends on that channel function, not just on the protein being present (channel-dead point mutants fail to rescue). Comparator check: proteins in the same role carry this term. The partner nuclear-envelope Ca2+ channels CNGC15A/CNGC15B and the downstream decoders CCaMK and CYCLOPS are annotated to GO:0009877 by IMP in Medicago/Lotus. Supporting Evidence: PMID:31420535 7a, plants expressing D442A, E493A, or E493Q mutants failed to form root nodules upon rhizobia infection demonstrating the importance of Ca2+ binding for the physiological function of CASTOR. PMID:15616514 Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. |
| GO:0036377 arbuscular mycorrhizal association | IMP PMID:15616514 Plastid proteins crucial for symbiotic fungal and bacterial ... | NEW | Summary: CASTOR is a common symbiosis gene. castor mutants (including castor-2, a pore mutant with altered channel properties) are defective in arbuscular mycorrhiza as well as root nodule symbiosis, and fungal entry into root epidermal cells is blocked. Reason: Same argument as for nodulation. CASTOR's channel activity is a step in the nuclear Ca2+ spiking used by both symbioses, so it participates in the process and is not just required for it. Comparator check: CNGC15A/CNGC15B and CYCLOPS carry GO:0036377 by IMP. Evidence that CASTOR is needed for the fungal symbiosis comes from the original mutant characterisation (abstract-only in cache). The Charpentier et al. full text restates that castor-2 is defective in both symbioses. Supporting Evidence: PMID:15616514 CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking PMID:19106374 The castor-2 mutant is defective in Ca 2+ spiking ( Figure 5 ) and both root nodule and arbuscular mycorrhiza symbioses ( Imaizumi-Anraku et al., 2005 ). |
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Download this section (compressed HTML)Q: What is the dominant ion carried by native full-length CASTOR in the nuclear envelope during symbiotic Ca2+ spiking: K+ as a counter-ion (bilayer data, PMID:19106374) or Ca2+ (truncated-construct patch clamp, PMID:31420535)? The answer decides whether potassium channel activity or calcium channel activity is the right MF term.
Q: Does CASTOR reside in the inner, outer or both nuclear membranes, and does its topology match the direction of K+ flux that the counter-ion model predicts?
Q: Does Lotus CASTOR physically associate with Lotus CNGC15 orthologs, as Medicago DMI1 does, and should that complex be captured as a GO-CAM with CASTOR and POLLUX as separate channel activities?
Experiment: Nuclear-envelope patch clamp (nucleus-attached or isolated nuclei) of wild-type, castor and castor-2 Lotus root cells under physiological ion gradients, to measure the native CASTOR-dependent current and its K+/Ca2+ selectivity.
Type: electrophysiology
Experiment: High-resolution immuno-EM or super-resolution imaging with membrane-preserving fixation to place CASTOR in the inner versus outer nuclear membrane.
Type: localization
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