ADHD meds and Catecholamine levels in the PFC

[Seppi]

I meant effluxing as in facilitated exchange, not channel mode. Did you read my post above?

I know what you meant. I'm stating there's no evidence that amphetamine, which is a DAT substrate, is subject to DAT uptake when DAT is effluxing/spewing out DAT substrates, regardless of whether or not DAT is in the channel/burst efflux configuration.

 

[serotonin2A]

Quote me then.
.

OK, I think I found the problem. You wrote earlier:

Amphetamine can not enter through an effluxing transporter, so I don't understand the point you're making. .

The miscommunication we are having here is (I think) because you are using "effluxing transporter" to describe DAT channel mode. That is indeed one way the transporter can efflux. But it isn't the only way. The transporter can also efflux by facilitated exchange, even if that isn't the primary way that it does woth amphetamine. No one in the field uses the terminology "effluxing mode" to describe that DAT state, and one reason is because it does not have a standardized meaning.

So writing the amphetamine cannot be taken up by DAT when it is in "effluxing mode" isn't entirely accurate. Amphetamine can't be taken up when dopamine efflux is occuring through DAT in channel mode.

 

[Seppi]

The miscommunication we are having here is (I think) because you are using "effluxing transporter" to describe DAT channel mode. That is indeed one way the transporter can efflux. But it isn't the only way. The transporter can also efflux by facilitated exchange, even if that isn't the primary way that it does woth amphetamine. No one in the field uses the terminology "effluxing mode" to describe that DAT state, and one reason is because it does not have a standardized meaning.


So writing the amphetamine cannot be taken up by DAT when it is in "effluxing mode" isn't entirely accurate. Amphetamine can't be taken up when dopamine efflux is occuring through DAT in channel mode.

DAT phosphorylation can put DAT in multiple configurations that produce efflux; it all depends on what residues are phosphorylated (NB: PKA [mediates internalization], PKC [mediates internalization and efflux], and CAMKII [mediates efflux] do not phosphorylate identical sets of residues). I'm not aware of any evidence that supports the facilitated exchange hypothesis, but if you know of a fairly recent review which covers evidence of it, I'd be very interested in reading it.

 

[Seppi]

Below is an excerpt that I placed on a WP talk page a while back which mentions various protein kinases involved in amphetamine-induced DAT phosphorylation and some of the residues they're known to phosphorylate in vitro:

• Wang Q, Bubula N, Brown J, Wang Y, Kondev V, Vezina P (May 2016). "PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux". Neurosci. Lett. 622: 78–82. doi:10.1016/j.neulet.2016.04.051. PMID 27113203.
  • "The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. ... The results of in vitro experiments using a recombinant N-terminal peptide of DAT [11,17] indicate that PKC phosphorylates the S4, S7, and S13 residues, that the S7 and S13 residues are also phosphorylated by PKA and CaMKII respectively, and that the T53 residue is phosphorylated by ERK1/2 (Fig. 1). ... Together, these findings suggest that PKC is not the only protein kinase that regulates amphetamine-induced DA efflux and, importantly, that it may function in concert with others at multiple residues in the N-terminal of the DAT to fully regulate its function. Indeed, DA efflux is regulated by several kinases in addition to PKC, including CaMKII and ERK1/2 [5,6], and all are capable of regulating the DAT by phosphorylating residues in its N-terminal [11–15,17] ... As some but not all findings indicate that CaMKII contributes to acute amphetamine-induced DA efflux and behaviors [12-14; cf,20], it remains possible that the inhibitory effect of the DAT-S13A mutant on DA efflux observed in the present study might in part reflect an action of CaMKII ... In addition, in the present experiments, S/T-A mutation of the non-PKC residue S12 and the ERK1/2 residue T53 were each found to reduce amphetamine-induced DA efflux by approximately 25% as well. ... Indeed, the lack of inhibition of amphetamine-induced DA efflux observed in the present study with the DAT-S7A mutant may reflect the integration at S7 of antagonistic signaling by PKC and PKA pathways as this residue is phosphorylated by both kinases [17]."
• Bermingham DP, Blakely RD (October 2016). "Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters". Pharmacol. Rev. 68 (4): 888–953. doi:10.1124/pr.115.012260. PMID 27591044.
  • "The Amara laboratory recently provided evidence that AMPH triggered DAT endocytosis is clathrin-independent and requires the small GTPase Rho (Wheeler et al., 2015), which mediates another dynamin-dependent mode of endocytosis (Croise et al., 2014). These lines of evidence are consistent with a PKC-independent mode of DAT internalization by AMPH. ... Recent work from the Amara laboratory has implicated PKA signaling in the regulation of Rho-mediated DAT internalization, specifically in response to AMPH treatment (Wheeler et al., 2015). ...
    Whereas little support for CaMKII regulation of DA uptake exists, substantial evidence supports a role for the kinase in DAT-dependent DA efflux triggered by AMPH or DAT mutations. ... Importantly, AMPH treatment of DAT transfected cells produced a rise in intracellular Ca2+ that could be blocked by thapsigargin or cocaine, supporting a model whereby AMPH is first transported into cells where it can then produce release of endoplasmic reticulum Ca2+ stores. Subsequently, AMPH was shown to activate CaMKII in DAT transfected cells (Wei et al., 2007). ... As noted above, an important role for CaMKII activity in AMPH-evoked DA efflux has been defined through the use of organic and peptide CaMKII inhibitors, intracellular kinase perfusion and the use of CaMKII KO/knock-in mouse models. The question naturally arises as to whether this contribution arises from direct, CaMKII-mediated DAT phosphorylation. ... At present, information is lacking as to the site(s) that support CaMKII phosphorylation of DAT in vivo ... The current model for how CaMKII participates in AMPH-triggered DA efflux involves binding of the kinase to the transporter C terminus followed by phosphorylation of one or more Ser residues in the transporter N terminus. This phosphorylation is then thought to facilitate conformational changes that place the transporter in a “DA efflux-willing” conformation. ...
    Thus, Kantor et al. (2004) described enhancement of DA efflux by PC-12 cells that was dependent on external Ca2+ and is blocked by the voltage-gated Ca2+ channel (VGCC) inhibitors v-conotoxin and nifedipine. The reader will recall that evidence suggests that DAT-mediated DA efflux after AMPH treatment relies more on intracellular Ca2+ stores than extracellular Ca2+, ... Interestingly, AMPH also elicited a greater increase in Ca2+ elevations after repeated treatment, suggesting possible changes in expression/activity of Ca2+ channels as well. The actions of AMPH to elevate Ca2+ levels were blocked by desipramine, suggesting that AMPH-induced depolarization may be responsible for Ca2+ channel activation. Consistent with this idea, Cameron et al. (2015) recently reported an ability of AMPH to activate VGCCs via transporter-mediated depolarization. ...
    Based on work from Chen et al. (2013) that showed that PKCβ appears to function upstream of ERK1/2, which are strong candidates for targeting Thr53 (Gorentla et al., 2009), it is possible that PKCβ may act through ERK1/2 to increase Thr53 phosphorylation and therefore positively regulate DAT, whereas other PKC isoforms act to downregulate DAT activity, potentially through direct phosphorylation of the transporter or other interacting proteins. ... As an equivalent loss of AMPH evoked efflux capacity was observed for both Ala and Asp substitutions, the precise role of phosphorylation at Thr53 in AMPH-induced DA efflux remains to be established."

 

[serotonin2A]

DAT phosphorylation can put DAT in multiple configurations that produce efflux; it all depends on what residues are phosphorylated (NB: PKA [mediates internalization], PKC [mediates internalization and efflux], and CAMKII [mediates efflux] do not phosphorylate identical sets of residues). I'm not aware of any evidence that supports the facilitated exchange hypothesis, but if you know of a fairly recent review which covers evidence of it, I'd be very interested in reading it.

The facilitated exchange model was the dominant explanation for amphetamine-induced dopamine release in the 1980s and 1990s. Most reviews still include it as part of the mechanism of action of amphetamine. For example, a 2009 review wrote (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729543/): "These studies suggest that while facilitated exchange diffusion may contribute to AMPH-mediated monoamine release, it cannot account for all experimental observations to date."

I'm a little surprised that you would say that you are not aware of any evidence that supports the facilitated exchange hypothesis, because any review of amphetamine action lists the evidence. For example, the same review from 2009:

"Evidence in support of this model of AMPH-induced efflux demonstrates that AMPH accumulation in rat synaptosomes is saturable, temperature-dependent, and ouabain-sensitive, implicating an active transport mechanism for AMPH³⁵. Additional evidence for an active transport mechanism has been supported by several electrophysiology studies illustrating AMPH’s ability to generate DA-like transporter associated currents."

The way that passage was written, the author is concluding that evidence exists that is consistent with the model. I'm not saying that I agree with the model, but I am aware of it, and I don't think there is enough evidence to discard it. And until we can completely disregard this potential aspect of amphetamine's action, calling the channel mode the efflux mode is going to generate confusion.

 

[Seppi]

The facilitated exchange model was the dominant explanation for amphetamine-induced dopamine release in the 1980s and 1990s. Most reviews still include it as part of the mechanism of action of amphetamine. For example, a 2009 review wrote (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729543/): "These studies suggest that while facilitated exchange diffusion may contribute to AMPH-mediated monoamine release, it cannot account for all experimental observations to date."

I'm a little surprised that you would say that you are not aware of any evidence that supports the facilitated exchange hypothesis, because any review of amphetamine action lists the evidence. For example, the same review from 2009:

"Evidence in support of this model of AMPH-induced efflux demonstrates that AMPH accumulation in rat synaptosomes is saturable, temperature-dependent, and ouabain-sensitive, implicating an active transport mechanism for AMPH³⁵. Additional evidence for an active transport mechanism has been supported by several electrophysiology studies illustrating AMPH’s ability to generate DA-like transporter associated currents."

The way that passage was written, the author is concluding that evidence exists that is consistent with the model. I'm not saying that I agree with the model, but I am aware of it, and I don't think there is enough evidence to discard it. And until we can completely disregard this potential aspect of amphetamine's action, calling the channel mode the efflux mode is going to generate confusion.

Interesting. Thanks.