Several transporters which belong to the SLC6 gene family are likely to accept GCs as substrates (Table
1). The SLC6 members are Na
+- and Cl
--dependent transporters for neurotransmitters, amino acids and osmolytes, including GABA, betaine, taurine, and CT [
47]. CRT mediates transport of CT, GAA, and CTN, with Km values of 46 μM/29 μM [
48,
49], 269 μM/412 μM [
50], and 52 mM [
51], respectively. The transport affinities of GAA and CTN for CRT are one and three orders of magnitude lower than that of CT, respectively. CT competitively inhibits CRT-mediated GAA transport with a Ki value of 60.5 μM [
50]. Therefore, it is necessary to consider the physiological fluid concentrations of CT and GAA in order to evaluate the actual contribution of CRT to GAA transport. Since CRT is an electrogenic transporter, CT induces an inward current in
Xenopus oocytes expressing human CRT at a holding potential of -60 mV [
52]. GAA, GPA or GBA at the concentration of 1 mM induces significant inward current in CRT-expressing, but not water-injected, oocytes [
50]. In the inhibition study, CRT-mediated GAA transport was significantly inhibited by GES, but not by taurine or GABA [
50]. PCT and CTN induce inward current to a lesser extent in CRT-expressing oocytes, while GSA and MG do not induce inward current [
50]. Thus, GPA, GBA and GES are potent substrates for CRT, whereas CRT does not recognize GSA, MG, taurine or GABA as a substrate. Dodd and Christie [
53] have reported that two or three amino acid substitutions result in the loss of CT transporter activity and gain of a specific GABA transporter function. Because GPA inhibits mouse GABA transporter 3 and 4 (GAT3/SLC6A13 and GAT4/SLC6A11) [
54,
55], there is a possibility that mouse GAT3 and GAT4 mediate transport of GCs. Therefore, these amino acid residues may be involved in GAT-mediated transport of GCs which are not CRT substrates. TauT accepts taurine, GAA, and GABA as substrates with Km values of 43 μM [
46], 215 μM [
56] and 1.46 mM [
57], respectively. The transport affinities of GAA and GABA for TauT are approximately one and two orders of magnitude lower than that of taurine, respectively. TauT-mediated GAA uptake is significantly inhibited by taurine, GABA, GPA, GBA and GES, whereas CT, GSA, MG and CTN have no effect [
56]. This result suggests that GPA, GBA and GES are good substrates for TauT, whereas CT, GSA, MG, and CTN are not.
Table 1
Inhibitory effect of various compounds on [14C]GAA uptake by HEK293 cells stably overexpressing CRT (CRT/HEK293 cells) and Xenopus oocytes expressing CRT (CRT/oocytes) or TauT (TauT/oocytes), and [14C]CTN uptake by Xenopus oocytes expressing rOCT3(rOCT3/oocytes).
Control | 100 ± 4 | 100 ± 9 | 100 ± 9 | 100 ± 19 |
Guanidinoacetate (GAA) | 19.2 ± 2.6* | 44.6 ± 2.7* | 53.2 ± 9.6* | - |
β-Guanidinopropionate (GPA) | 1.81 ± 0.14* | 0.944 ± 0.189* | 4.77 ± 0.66* | 143 ± 21 |
γ-Guanidinobutyrate (GBA) | 10.0 ± 0.6* | 21.4 ± 2.0* | 21.7 ± 4.2* | - |
Guanidinoethansulfonate (GES) | 23.1 ± 1.1* | - | 4.66 ± 0.22* | - |
Guanidinosuccinate (GSA) | 101 ± 1 | 107 ± 12 | 103 ± 10 | - |
Creatine (CT) | 3.06 ± 0.24* | 2.91 ± 0.23* | 98.4 ± 12.7 | 137 ± 24 |
Taurine | 111 ± 5 | - | 3.00 ± 0.19* | - |
γ-Aminobutyric acid (GABA) | 95.5 ± 2.9 | - | 34.0 ± 4.6* | - |
Creatinine (CTN) | 93.3 ± 5.3 | 123 ± 10 | 97.9 ± 14.6 | - |
Methylguanidine (MG) | 82.3 ± 15.2 | 126 ± 14 | 110 ± 18 | 47.2 ± 18.9** |
Creatine phosphate (PCT) | 93.4 ± 6.8 | 97.9 ± 14.1 | 117 ± 6 | - |
Guanidine | 114 ± 6 | 88.2 ± 13.4 | - | 137 ± 22 |
L-Arginine | 86.6 ± 6.3 | - | - | - |
Glycine | 126 ± 4 | - | - | - |
L-Alanine | 111 ± 3 | - | - | - |
L-Aspartic acid | 98.5 ± 2.2 | - | - | - |
Tetraethylammonium | 128 ± 29 | 82.8 ± 7.3 | - | 48.1 ± 8.3** |
Benzylpenicillin | 110 ± 14 | 115 ± 12 | - | - |
Rat organic cation transporter 3 (rOCT3/SLC22A3) and human OCT2 (hOCT2/SLC22A2) mediate low-affinity CTN transport with Km values of 47.7 mM [
51] and 4.0 mM [
58], respectively. In contrast, human OCT1 (hOCT1/SLC22A1) does not recognize CTN [
58]. MG inhibits rOCT3-mediated CTN transport [
51], implying that MG is also a substrate for rOCT3. Guanidine, GSA and MG inhibit hOCT1 and/or hOCT2-mediated tetraethylammonium uptake, while GAA has little effect on the uptake [
59]. Thus, the SLC22 members are likely to mediate transport of GSA, CTN, guanidine, and MG, which are not recognized by the SLC6 family members.