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  • The dopamine substrate currents IDA

    2022-01-15

    The dopamine substrate currents (IDA – IControl) were determined at different holding potentials (IV curves) in transfected HT22 cells (Fig. 5A). The plot for HT22 cells transfected with DAT exhibited the typical behavior of dopamine transporter currents (Ingram et al., 2002; Sonders et al., 1997), with the transport-associated depolarizing conductance increasing at more hyperpolarized potentials (i. e., for holding potentials more negative than −50 mV). However, in the presence of Kv7.2/7.3 potassium channels (Fig. 5A, open circle symbols), DAT-associated currents were almost negligible between −110 and −60 mV. As Kv7.2/7.3 channels activate within subthreshold potentials (∼−60 mV) (Brown and Passmore, 2009; Wang et al., 1998), we did not expect to see any contribution to the whole cell conductance at those hyperpolarized potentials (<−60 mV). Nevertheless, the close association between DAT and Kv7.2/Kv7.3 suggested by the previously mentioned results could offer an explanation, wherein the depolarizing current produced by DAT activation would influence the M-channels in close proximity, and the potassium efflux promoted by opening of these channels could completely counteract the dopamine depolarizing effect. Indeed, the inward directed, depolarizing Na+ current consequent of DAT activity was so efficiently opposed by YFP-Kv7.2/7.3 that the IV curve in the control condition without DAT (+YFP-Kv7.2/7.3; x symbols in Fig. 5A) was barely distinguishable from the IV for Ristocetin A sulfate expressing the transporter (+HA-DAT + YFP-Kv7.2/7.3; open circles in Fig. 5A). Dopamine by itself did not enhance the M-currents carried by Kv7.2/7.3 channels (Fig. 5B). Rather the opposite, the effect of dopamine by itself on YFP-Kv7.2/7.3 channels was inhibitory, mainly at potentials above −50 mV (see discussion). Similarly, glutamate substrate currents (IGlu – IControl) were measured in transfected HT22 cells (Fig. 6A), in the absence or presence of XE-991 to better assess the contribution of the Kv7 channels to GLT-1 activity. As expected, cells transfected with HA-GLT-1 (Fig. 6A, closed squares) showed a depolarizing current which was increased at more hyperpolarized potentials. As with dopamine and DAT, the GLT-1 substrate current at hyperpolarized potentials was significantly reduced in the presence of YFP-Kv7.2/7.3 (Fig. 6A, open circles), suggesting that the depolarization induced by glutamate transport was quickly neutralized by potassium efflux thanks to M-current activation. This observation is consistent with the blockade of the stimulatory effect of radiolabelled uptake in the presence of XE-991. The effect of XE-991 is unlikely to be mediated by a direct inhibition of GLT-1 since electrophysiological recordings in control cells transfected only with GLT-1 did not reveal any effect of XE-991 on the I/V relationships. Finally, control cells transfected only with YFP-Kv7.2/7.3 (Fig. 6A, x symbols) did not show a significant glutamate-associated current, and glutamate by itself did not affect the YFP-Kv7.2/7.3 currents (Fig. 6B). We blocked M-channels with 20 μM XE-991 in cells transfected with HA-GLT-1 + YFP-Kv7.2/7.3 in the absence and in the presence of glutamate (Fig. 7A), unmasking their effect on GLT-1 substrate current. The apparent GLT-1 current was bigger in the presence of the XE-991, mainly at more negative membrane potentials (see Fig. 7A and the inset). We were also able to measure the XE-991-sensitive (Kv7.2/7.3) current in control conditions after adding 1 mM glutamate (Fig. 7B). The IV curves and the bar plot demonstrate that in the presence of glutamate and GLT-1, M-channels were active even at −50 mV (Fig. 7B, histogram inset) and more negative potentials, again probably because activation of GLT-1 transporters (tightly coupled to Kv7.2/7.3 channels) produced an inward Na+ current. Altogether, these data support the existence of a tight coupling and crosstalk between GLT-1 and the M-channels that might prevent neuronal depolarization after the release of glutamate, favoring operation of the electrogenic transporter.