Alternative titles; symbols
HGNC Approved Gene Symbol: SLC12A5
Cytogenetic location: 20q13.12 Genomic coordinates (GRCh38) : 20:46,021,686-46,060,150 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 20q13.12 | {Epilepsy, idiopathic generalized, susceptibility to, 14} | 616685 | Autosomal dominant | 3 |
| Developmental and epileptic encephalopathy 34 | 616645 | Autosomal recessive | 3 |
The SLC12A5 gene encodes the neuronal KCC2 channel that is the major extruder of intracellular chloride in mature neurons. In the presence of low intraneuronal chloride, the binding of GABA and glycine to their ionotropic receptors results in chloride influx with subsequent hyperpolarization contributing to neuronal inhibition. The SLC12A5 gene is exclusively expressed in the central nervous system (summary by Stodberg et al., 2015).
The cation-chloride cotransporter gene family includes that Na-Cl cotransporter (NCC; 600968), the Na-K-2Cl cotransporters (NKCCs; see 600839), and the K-Cl cotransporters (KCCs). KCCs normally lower intracellular chloride concentrations below the electrochemical equilibrium potential. Depending on the chemical concentration gradients of potassium and chloride, KCC2 can operate as a net efflux or influx pathway (Hubner et al., 2001).
Payne et al. (1996) cloned Slc12a5, which they called Kcc2, from a rat brain cDNA library. In situ hybridization studies demonstrated that rat Kcc2 is expressed at high levels in neurons throughout the central nervous system, including CA1-CA4 pyramidal neurons of the hippocampus, granular cells and Purkinje neurons of the cerebellum, and many groups of neurons throughout the brainstem. Using in situ hybridization, Hubner et al. (2001) detected murine Kcc2 in the ventral horns of the spinal cord at embryonic day 12.5 and throughout the spinal cord at birth. Immunofluorescence revealed Kcc2 protein localized at inhibitory synapses of the spinal cord.
By screening for cDNAs with the potential to encode large proteins expressed in brain, Hirosawa et al. (1999) identified a cDNA encoding SLC12A5, which they called KIAA1176. The predicted 1,101-amino acid protein is 99% identical to rat Kcc2. RT-PCR analysis detected high expression of KIAA1176 in all brain regions examined. Except for moderate levels in pancreas, KIAA1176 expression was very low or undetectable in all other tissues tested.
Using antisense oligonucleotide experiments, Rivera et al. (1999) showed that inhibition of Kcc2 expression produced a marked positive shift in the reversal potential of GABA-A (see 137192) responses in functionally mature rat hippocampal pyramidal neurons. They concluded that KCC2 is the main chloride extruder to promote fast hyperpolarizing postsynaptic inhibition in the brain.
Szabadics et al. (2006) found that KCC2 is present in the human cortex but absent from the axo-axonic cells. They found that axo-axonic cells can depolarize pyramidal cells and can initiate stereotyped series of synaptic events in rat and human cortical networks because of the depolarized reversal potential for axonal relative to perisomatic GABAergic inputs.
Using radiation hybrid analysis, Hirosawa et al. (1999) mapped the SLC12A5 gene to chromosome 20. By FISH, Sallinen et al. (2001) mapped the human SLC12A5 gene to chromosome 20q12-q13.1 and the mouse gene to chromosome 5.
Developmental and Epileptic Encephalopathy 34
In 4 children from 2 unrelated families with developmental and epileptic encephalopathy-34 (DEE34; 616645), Stodberg et al. (2015) identified compound heterozygous or homozygous missense mutations in the SLC12A5 gene (606726.0001-606726.0003). The mutations were found by exome sequencing and segregated with the disorder in the families. In vitro functional expression studies showed that the mutations caused decreased membrane expression of SLC12A5, impaired posttranslational modification, and a loss of transporter function, resulting in impaired normal synaptic inhibition and promotion of neuronal excitability.
Idiopathic Generalized Epilepsy 14
Kahle et al. (2014) identified 2 different heterozygous missense variants in the SLC12A5 gene (R952H, 606726.0004 and R1049C, 606726.0005) that were enriched among individuals of French Canadian origin with idiopathic generalized epilepsy-14 (EIG14; 616685) compared to controls. In vitro functional expression studies showed that the variants impaired chloride extrusion capacities, resulting in less hyperpolarized glycine equilibrium potentials, and also impaired stimulatory phosphorylation at residue ser940, a key regulatory site of channel function. The overall effect impaired the function of SLC12A5. The variants were inherited from an unaffected parent in several cases, consistent with incomplete penetrance. The authors used a targeted DNA-sequencing approach to screen the cytoplasmic C-terminal region of SLC12A5 which is an important regulatory region of transporter function.
Using gene targeting, Hubner et al. (2001) generated mice lacking the Kcc2 gene. The Kcc2-deficient mice had severe motor deficits and died immediately after birth due to an inability to breathe. Sciatic nerve recordings revealed abnormal spontaneous electrical activity and altered spinal cord responses to peripheral electrical stimuli. Hubner et al. (2001) concluded that disruption of Kcc2 interferes with the function of alpha motoneurons. Using patch-clamp measurements of embryonic spinal cord motoneurons, they demonstrated an excitatory action of both GABA and glycine in the absence of Kcc2 but not in its presence, revealing a crucial role of Kcc2 for synaptic inhibition.
Boulenguez et al. (2010) found that the expression of Slc12a5 was downregulated after spinal cord injury in rats, particularly in motoneuron membranes in the ventral horn. This resulted in a depolarization of the Cl- equilibrium potential and reduced the strength of postsynaptic inhibition, causing increased cell excitability. Blocking Slc12a5 in intact rats reduced the rate-dependent depression (RDD) of the Hoffmann reflex, as is observed in spasticity. RDD was also decreased in Slc12a6 (604878)-deficient mice and in intact rats after intrathecal BDNF (113505) administration, which downregulates Slc12a5. The early decrease in Slc12a5 after spinal cord injury could be prevented by sequestering BDNF at the time of spinal cord injury. Conversely, after spinal cord injury, BDNF upregulated Slc12a5 and restored rate-dependent depression. Boulenguez et al. (2010) suggested a role for SLC12A5 in spasticity that occurs from reduced inhibitory mechanisms after spinal cord injury.
Stodberg et al. (2015) found that complete knockout of 2 KCC2 orthologs in zebrafish resulted in early motor deficits characterized by jerky spasmodic movements.
In 2 sibs (family A), born of unrelated Swedish parents, with developmental and epileptic encephalopathy-34 (DEE34; 616645), Stodberg et al. (2015) identified compound heterozygous mutations in the SLC12A5 gene: a c.1277T-C transition (c.1277T-C, ENST00000454036) in exon 9, resulting in a leu426-to-pro (L426P) substitution, and a c.1652G-A transition in exon 13, resulting in gly551-to-asp (G551D; 606726.0002) substitution. Both mutations occurred at highly conserved residues: L426P in transmembrane domain 6 (TM6) and G551D in the intracellular loop between TM8 and TM9. The mutations, which were found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family and were not found in the dbSNP (build 137), 1000 Genomes Project, or Exome Sequencing Project databases. Transfection of the mutations into HEK293 cells showed that both caused depolarized chloride extrusion compared to wildtype, consistent with passive equilibration, intracellular chloride accumulation, and a near complete loss of function. In addition, both mutant proteins had decreased cell surface expression and impaired posttranslational modification compared to wildtype. The patients presented with refractory seizures at 3 to 4 months of age. The phenotype was consistent with EIMFS.
For discussion of the c.1652G-A transition (c.1652G-A, ENST00000454036) resulting in a gly551-to-asp (G551D) substitution in the SLC12A5 gene that was found in compound heterozygous state in a patient with developmental and epileptic encephalopathy-34 (DEE34; 616645) by Stodberg et al. (2015), see 606726.0001.
In 2 sibs (family B), born of consanguineous Pakistani parents, with developmental and epileptic encephalopathy-34 (DEE34; 616645), Stodberg et al. (2015) identified a homozygous c.932T-A transversion (c.932T-A, ENST00000454036) in exon 8 of the SLC12A5 gene, resulting in a leu311-to-his (L311H) substitution at a highly conserved residue in the extracellular loop between TM5 and TM6. The mutation, which was found by a combination of autozygosity mapping and exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family, and was not found in the dbSNP (build 137), 1000 Genomes Project, or Exome Sequencing Project databases. Transfection of the mutation into HEK293 cells showed that it caused depolarized chloride extrusion compared to wildtype, consistent with passive equilibration, intracellular chloride accumulation, and a partial loss of function. The mutant protein also showed decreased cell surface expression and impaired posttranslational modification compared to wildtype. The patients presented with migrating focal seizures at 6 to 10 weeks of life.
In 5 patients of French Canadian origin with idiopathic generalized epilepsy-14 (EIG14; 616685), Kahle et al. (2014) identified a heterozygous c.2855G-A transition (c.2855G-A, NM_020708.4) in the SLC12A5 gene, resulting in an arg952-to-his (R952H) substitution at a conserved residue in the cytoplasmic C-terminal domain. The variant was found in 5 of 380 patients with EIG (allele frequency of 0.66%) and in 5 of 1,214 controls (allele frequency of 0.21%), yielding an odds ratio (OR) of 3.21 for development of EIG (p = 0.065). Parental DNA was available from 3 of the patients, and the variant was inherited from an unaffected parent in all 3 cases, indicating incomplete penetrance. In vitro cellular functional studies indicated that the expression of the mutation resulted in a decreased hyperpolarizing shift (less hyperpolarized) in the reversal potential of glycine receptors, as well as decreased efficacy in chloride extrusion compared to wildtype. These changes resulted in a higher basal level of intracellular chloride levels and less hyperpolarized responses to glycine, and there was evidence for a dominant-negative effect. Cultured hippocampal neurons expressing the mutation showed a more than 2-fold decrease in mutant protein expression compared to wildtype. Additional studies indicated that the mutant protein had a significant decrease in stimulatory phosphorylation at residue S940, which could explain the impaired function.
Puskarjov et al. (2014) identified a heterozygous R952H variant in the SLC12A5 gene in 3 affected members of an Australian family with febrile seizures. Segregation of the variant (rs142740233) in this kindred was difficult because of uncertain phenotyping, but there was some evidence of incomplete penetrance. The variant was found at a low frequency (0.07%) in public databases (dbSNP, 1000 Genomes Project, and Exome Variant Server). In vitro functional expression studies showed markedly decreased surface expression of the mutant protein (61% of wildtype) as well as decreased chloride extrusion compared to wildtype. Thus, the mutant protein was associated with deficits in maintaining the chloride driving force required for hyperpolarizing GABA-mediated responses. Transfection of the variant into rats and into mouse cortical neurons showed that it compromised dendritic spine formation and maturation. Puskarjov et al. (2014) suggested that the decrease in SLC12A5-dependent hyperpolarizing inhibition would promote triggering of seizures, and that decreased dendritic spine formation could lead to desynchronization of overall excitability, which may also contribute to seizures.
Lek et al. (2016) questioned the pathogenicity of this variant because it has a high allele frequency (0.0186) in the South Asian population in the ExAC database.
In 3 patients of French Canadian origin with idiopathic generalized epilepsy-14 (EIG14; 616685), Kahle et al. (2014) identified a heterozygous c.3145C-T transition (c.3145C-T, NM_020708.4) in the SLC12A5 gene, resulting in an arg1049-to-cys (R1049C) substitution at a highly conserved residue in the cytoplasmic C-terminal domain. The variant was found in 3 of 380 patients with EIG (allele frequency of 0.39) and in 1 of 1,214 controls (allele frequency of 4.12 x 10(-4)), yielding an odds ratio (OR) of 9.61 (p = 0.044). In vitro cellular functional studies indicated that the expression of the mutation resulted in a decreased hyperpolarizing shift (less hyperpolarized) in the reversal potential of glycine receptors, as well as decreased efficacy in chloride extrusion compared to wildtype. These changes resulted in a higher basal level of intracellular chloride levels and less hyperpolarized responses to glycine. Cultured hippocampal neurons expressing the mutation showed normal membrane localization similar to wildtype. Additional studies indicated that the mutant protein had a significant decrease in stimulatory phosphorylation at residue S940, which could explain the impaired function.
Boulenguez, P., Liabeuf, S., Bos, R., Bras, H., Jean-Xavier, C., Brocard, C., Stil, A., Darbon, P., Cattaert, D., Delpire, E., Marsala, M., Vinay, L. Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury. Nature Med. 16: 302-307, 2010. [PubMed: 20190766] [Full Text: https://doi.org/10.1038/nm.2107]
Hirosawa, M., Nagase, T., Ishikawa, K., Kikuno, R., Nomura, N., Ohara, O. Characterization of cDNA clones selected by the GeneMark analysis from size-fractionated cDNA libraries from human brain. DNA Res. 6: 329-336, 1999. [PubMed: 10574461] [Full Text: https://doi.org/10.1093/dnares/6.5.329]
Hubner, C. A., Stein, V., Hermans-Borgmeyer, I., Meyer, T., Ballanyi, K., Jentsch, T. J. Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. Neuron 30: 515-524, 2001. [PubMed: 11395011] [Full Text: https://doi.org/10.1016/s0896-6273(01)00297-5]
Kahle, K. T., Merner, N. D., Friedel, P., Silayeva, L., Liang, B., Khanna, A., Shang, Y., Lachance-Touchette, P., Bourassa, C., Levert, A., Dion, P. A., Walcott, B., and 11 others. Genetically encoded impairment of neuronal KCC2 cotransporter function in human idiopathic generalized epilepsy. EMBO Rep. 15: 766-774, 2014. [PubMed: 24928908] [Full Text: https://doi.org/10.15252/embr.201438840]
Lek, M., Karczewski, K. J., Minikel, E. V., Samocha, K. E., Banks, E., Fennell, T., O'Donnell-Luria, A. H., Ware, J. S., Hill, A. J., Cummings, B. B., Tukiainen, T., Birnbaum, D. P., and 68 others. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536: 285-291, 2016. [PubMed: 27535533] [Full Text: https://doi.org/10.1038/nature19057]
Payne, J. A., Stevenson, T. J., Donaldson, L. F. Molecular characterization of a putative K-Cl cotransporter in rat brain: a neuronal-specific isoform. J. Biol. Chem. 271: 16245-16252, 1996. [PubMed: 8663311] [Full Text: https://doi.org/10.1074/jbc.271.27.16245]
Puskarjov, M., Seja, P., Heron, S. E., Williams, T. C., Ahmad, F., Iona, X., Oliver, K. L., Grinton, B. E., Vutskits, L., Scheffer, I. E., Petrou, S., Blaesse, P., Dibbens, L. M., Berkovic, S. F., Kaila, K. A variant of KCC2 from patients with febrile seizures impairs neuronal Cl- extrusion and dendritic spine formation. EMBO Rep. 15: 723-729, 2014. [PubMed: 24668262] [Full Text: https://doi.org/10.1002/embr.201438749]
Rivera, C., Voipio, J., Payne, J. A., Ruusuvuori, E., Lahtinen, H., Lamsa, K., Pirvola, U., Saarma, M., Kaila, K. The K(+)/Cl(-) co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397: 251-255, 1999. [PubMed: 9930699] [Full Text: https://doi.org/10.1038/16697]
Sallinen, R., Tornberg, J., Putkiranta, M., Horelli-Kuitunen, N., Airaksinen, M. S., Wessman, M. Chromosomal localization of SLC12A5/Slc12a5, the human and mouse genes for the neuron-specific K(+)-Cl(-) cotransporter (KCC2) defines a new region of conserved homology. Cytogenet. Cell Genet. 94: 67-70, 2001. Note: Erratum: Cytogenet. Genome Res. 98: 232 only, 2002. [PubMed: 11701957] [Full Text: https://doi.org/10.1159/000048785]
Stodberg, T., McTague, A., Ruiz, A. J., Hirata, H., Zhen, J., Long, P., Farabella, I., Meyer, E., Kawahara, A., Vassallo, G., Stivaros, S. M., Bjursell, M. K. Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures. Nature Commun. 6: 8038, 2015. Note: Electronic Article. [PubMed: 26333769] [Full Text: https://doi.org/10.1038/ncomms9038]
Szabadics, J., Varga, C., Molnar, G., Olah, S., Barzo, P., Tamas, G. Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science 311: 233-235, 2006. [PubMed: 16410524] [Full Text: https://doi.org/10.1126/science.1121325]