Plasma Membrane Na⁺-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy

Abstract

SLC13A5 is a Na⁺-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na⁺:citrate^3- stoichiometry of 4:1. In humans, the Michaelis constant for SLC13A5 to transport citrate is ~600 μM, which is physiologically relevant given that the normal concentration of citrate in plasma is in the range of 150-200 μM. Li⁺ stimulates the transport function of human SLC13A5 at concentrations that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ~30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metabolism in the sperm mitochondria to generate biological energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metabolism. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to experimentally induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clinical consequences of the loss of function of this transporter between humans and mice. The markedly different biochemical features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact molecular mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-d-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.

Keywords: CIC (SLC25A1); GABA; NMDA receptor; NaCT (SLC13A5); cholesterol synthesis; citrate transporter; cytoplasmic citrate; fatty acid synthesis; mitochondrial citrate; neurotransmitters; tricarboxylic acid cycle; zinc.

Figure 1

Roles of the mitochondrial citrate transporter SLC25A1 and the plasma membrane citrate transporter SLC13A5 as the determinants of citrate levels in the mitochondrial matrix and the cytoplasm. NaCT, Na⁺-coupled citrate transporter; CIC, citrate carrier; CoA, coenzyme A; TCA, tricarboxylic acid cycle; IMM, inner mitochondrial membrane.

Figure 2

Functions of citrate in the cytoplasm of neurons. The enzymes listed are: 1: ATP:citrate lyase; 2: Acetyl CoA carboxylase; 3: Fatty acid synthase complex; 4: Thiolase; 5: HMG CoA synthase; 6: Aconitase; 7: Isocitrate dehydrogenase; 8: Transaminase; 9: Glutamate decarboxylase; 10: Choline acetyl transferase; 11: Protein (histone) acetyl transferases. CoA, coenzyme A; OAA, oxaloacetate; AA, amino acid; α-KA, α-Ketoacid; GABA, γ-aminobutyrate; HMG CoA, 3-hydroxy-3-methyl glutaryl CoA.

Figure 3

The possible role of zinc and NMDA receptors in loss-of-function SLC13A5 mutations. (A) normal condition. Astrocytes release citrate (yellow U-shaped symbols) into the extracellular space via an unknown mechanism (denoted by a question mark). The extracellular zinc binding site on the NR2A subunit (red) of the NMDA receptor is occupied by zinc ions (black filled circles), acting as a check on NMDA receptor-induced calcium influx. The plasma membrane citrate transporter NaCT (SLC13A5, pink) limits the accumulation of extracellular citrate by transporting citrate into the cytoplasm. Citrate extrusion from the mitochondria via the mitochondrial citrate carrier (CIC or SLC25A1, green) is also a source of cytosolic citrate; (B) When SLC13A5 function is lost, intracellular citrate levels are likely reduced while extracellular citrate levels rise to pathologically high levels. Citrate chelation of free zinc reduces the availability of zinc for the NMDA receptor, relieving the negative allosteric effect of zinc on NMDA receptor function, thereby increasing calcium flux through the NMDA receptor (note larger arrow indicating enhanced flux of calcium through the NMDA receptor). Schematics in A and B were motivated from [30,61]; (C) kinetic model simulations [55,62] of NMDA receptor currents generated by a 5 s pulse of glutamate under normal conditions (i.e., with free zinc) and under conditions in which extracellular citrate has chelated free zinc (i.e., without free zinc). Citrate chelation induces a larger and more sustained NMDA receptor current; (D) response of the kinetic model to six synaptic-like 1-ms pulses of glutamate, simulating a burst of gluatmatergic synaptic activity. Note that NMDA receptor-mediated currents summate and deactivate more slowly under conditions of elevated extracellular citrate levels than under the normal conditions. These simulations demonstrate that increased extracellular citrate levels have the potential to enhance NMDA receptor-mediated synaptic transmission, which is likely to yield pro-convulsant effects.