Bidirectional transport of 2-chloroadenosine by equilibrative nucleoside transporter 4 (hENT4): Evidence for allosteric kinetics at acidic pH

Abstract

Adenosine has been reported to be transported by equilibrative nucleoside transporter 4 (ENT4), encoded by the SLC29A4 gene, in an acidic pH-dependent manner. This makes hENT4 of interest as a therapeutic target in acidic pathologies where adenosine is protective (e.g. vascular ischaemia). We examined the pH-sensitivity of nucleoside influx and efflux by hENT4 using a recombinant transfection model that lacks the confounding influences of other nucleoside transporters (PK15-NTD). We established that [3H]2-chloroadenosine, which is resistant to metabolism by adenosine deaminase, is a substrate for hENT4. Transport of [3H]2-chloroadenosine at a pH of 6.0 in PK15-NTD cells stably transfected with SLC29A4 was biphasic, with a low capacity (Vmax ~ 30 pmol/mg/min) high-affinity component (Km ~ 50 µM) apparent at low substrate concentrations, which shifted to a high capacity (Vmax ~ 500 pmol/mg/min) low affinity system (Km > 600 µM) displaying positive cooperativity at concentrations above 200 µM. Only the low affinity component was observed at a neutral pH of 7.5 (Km ~ 2 mM). Efflux of [3H]2-chloroadenosine from these cells was also enhanced by more than 4-fold at an acidic pH. Enhanced influx and efflux of nucleosides by hENT4 under acidic conditions supports its potential as a therapeutic target in pathologies such as ischaemia-reperfusion injury.

Deletion of murine slc29a4 modifies vascular responses to adenosine and 5‐hydroxytryptamine in a sexually dimorphic manner

“…ENT4 differs from ENTs encoded by other members of the SLC29 family (ENT1, ENT2, ENT3) in that it also transports monoamines such as 5‐hydroxytryptamine (5‐HT; Engel, Zhou, & Wang, 2004; Shirasaka et al, 2017; Wang, 2016). Furthermore, it has a more restricted specificity for nucleosides than other ENTs, accepting only adenosine and some adenosine analogues (Tandio, Vilas, & Hammond, 2019) as substrates. There have been numerous studies highlighting the role of ENT4 in modulating the effects of 5‐HT in the CNS, but relatively few studies have examined its role in peripheral systems.…”

“…Since both adenosine and 5‐HT have well‐documented cardiovascular effects (Fidalgo, Ivanov, & Wood, 2013; McIntosh & Lasley, 2012; Sousa & Diniz, 2017; Watts, 2016), ENT4 may play a role in regulating the effects of these agents in the vasculature. A unique aspect of ENT4 is the enhancement of substrate flux in acidic conditions, such as those associated with vascular ischemia‐reperfusion injury (Barnes et al, 2006; Tandio et al, 2019; Zhou, Duan, Engel, Xia, & Wang, 2010). ENT4 has been implicated in regulating 5‐HT levels in rat heart, particularly during the reperfusion stage of ischemia‐reperfusion (Sonobe, Akiyama, Du, & Pearson, 2019), and modulation of adenosine actions in the vasculature has long been proposed as a therapy to attenuate ischemia‐reperfusion injury (Abd‐Elfattah, Aly, Hanan, & Wechsler, 2012; Abd‐Elfattah et al, 2013; Hirai & Ashraf, 1998; Rose et al, 2010; Van Belle, 1995; Yang & Leung, 2015).…”

Impact of SLC43A3/ENBT1 Expression and Function on 6-Mercaptopurine Transport and Cytotoxicity in Human Acute Lymphoblastic Leukemia Cells

“…This finding is congruent with our correlation data that shows a strong negative association between SLC29A4 expression and the cytotoxicity of 6-MP in the cell lines examined (Figure 5B). SLC29A4 encodes for ENT4, an acidic pH-activated adenosine and monoamine transporter (Barnes, Dobrzynski et al 2006, Tandio, Vilas et al 2019). ENT4 does not transport nucleobases, and the role of ENT4 in lymphoblasts has not been investigated.…”

Review of Transporter Substrate, Inhibitor, and Inducer Characteristics of Cladribine

“…Substrate specificity of ENT4 is somewhat different as it transports monoamines and thus was also denoted plasma membrane monoamine transporter because of its ability to transport organic cations including biogenic amines, cationic therapeutics, and neurotoxins [36]. ENT4 has been also shown to transport adenosine [59], and more recently 2-chloro-adenosine in a pH-dependent manner [60]. ENT4 is most abundantly expressed in the brain, where it may play a role in the clearance of monoamine neurotransmitters [61,62].…”

“…Taken together, there is robust information that cladribine is a substrate of ENT1, and data from two different in vitro systems showing that cladribine is also transported by ENT2. Evidence of ENT4-mediated transport of adenosine [59] and 2-chloro-adenosine [60] suggests that ENT4 may be considered a reasonable candidate transporter for cladribine. As shown recently, JN-desmoplastic small round cell tumor 1 cells abundantly expressing ENT4 mRNA were more sensitive to the pH-dependent cytotoxic effect of cladribine than cells with lower ENT4 expression.…”