Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants - PubMed (original) (raw)

Concentration and voltage dependence of DA-evoked steady-state currents. Concentration-dependent current responses were measured in six oocytes using a voltage jump protocol (see Materials and Methods). To control for differing levels of hDAT expression between oocytes, _I_DA −_I_Control subtractions for each DA concentration and potential were normalized to the current elicited by 10 μ

m

DA at −120 mV in the same cell (mean, −17.9 nA; range, −7.9 to −43.5 nA). A, Nonlinear curve fitting of mean current amplitudes for each DA concentration (x, 0.1 μ

m

; ▪, 0.3 μ

m

; ▵, 1.0 μ

m

; ♦, 3.0 μ

m

; ⊞, 10 μ

m

; •, 30 μ

m

). Over the potential range −120 to −20 mV, DA-elicited currents displayed an exponential dependence on voltage (mean, _e_-fold per 67 mV, solid lines; range, _e_-fold per 55–75 mV). At more positive potentials, however, current amplitudes were better described as having a linear relation to membrane potential (broken lines).B, The DA concentration dependence of mean-normalized currents (○) was well fit by a simple Michaelis–Menten equation for individual potentials over the range −120 to −20 mV. Currents appeared to saturate with increasing [DA] and displayed _K_0.5 values of 1.9–2.9 μ

m

. C, At potentials greater than −20 mV, mean normalized current amplitudes (▪) were more poorly described by Michaelis–Menten kinetics as a function of concentration. In some oocyte batches, high DA concentrations affected an endogenous conductance (see Results), and therefore the 30 μ

m

points (⊞) were omitted from the curve fits. D, At each membrane potential tested, geometric means of_K_0.5 and CI95 values (error bars) were determined from affinity values derived individually for each of six oocytes (see Materials and Methods). The apparent affinities (_K_0.5) of DA for eliciting transport-associated current (•) and for blocking a leak current (▪) are displayed. The _K_0.5 for the transport-associated current (in the range of −120 to −20 mV) demonstrates little voltage sensitivity. For comparison, also plotted is the DA apparent substrate affinity (_K_T, ——; CI95 - - -) determined in uptake assays using six different batches of oocytes. These oocytes were not voltage-clamped, although other oocytes from the same batches displayed resting membrane potentials in the range of −15 to −45 mV.