To determine whether OTC is a transportable substrate for SLC5A8, we used the
X. laevis expression system. SLC5A8 is an electrogenic transporter, and transport of monocarboxylate substrates via this transporter occurs with a Na
+:substrate stoichiometry of 2:1.
27,28 This makes the transport process electrogenic, with a net positive charge entering the cells during the cotransport of Na
+ and the monocarboxylate substrate via the transporter. This results in membrane depolarization that can be detected as inward currents under voltage-clamp conditions. With this rationale, we expressed human SLC5A8 in
X. laevis oocytes and monitored its transport function by electrophysiological means. Water-injected oocytes served as negative controls. Nicotinate was used as a positive control, because we have shown previously that exposure of SLC5A8-expressing oocytes to this monocarboxylate induces Na
+-dependent inward currents under voltage-clamp conditions.
36 Exposure of human SLC5A8-expressing oocytes to 1 mM nicotinate induced marked inward currents in the presence of Na
+ (
Fig. 2A). Such currents were not detectable in SLC5A8-expressing oocytes in the absence of Na
+, nor in water-injected oocytes (data not shown). We then examined whether OTC was recognized as a substrate by human SLC5A8 by monitoring the inward currents in human SLC5A8-expressing oocytes on exposure to this compound in the presence of Na
+ (
Fig. 2B). At a concentration of 1 mM, OTC induced inward currents in SLC5A8-expressing oocytes. These currents were not detectable in water-injected oocytes (data not shown). As seen with nicotinate, the currents induced by OTC were obligatorily dependent on the presence of Na
+. Similar results were obtained with three different oocytes. These data show that OTC is indeed a transportable substrate for SLC5A8 and that the transport process is Na
+-coupled and electrogenic.