Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 μM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na+-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at −90 mV). Applying step changes to the transmembrane potential, Vm, of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a τ of ∼15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<−40 mV) and activation at positive Vm (>0 mV). A similar inhibitory effect at Vm < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-Vm curve. Jumping the glutamate concentration to 100 μM generated biphasic, saturable transient transport and anion currents (Km ∼ 5 μM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1–3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.
Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on the transporter function. To do so, we coexpressed EAAC1WT with the mutant transporter EAAC1R446Q, which transports glutamine but not glutamate. Application of 50 μM glutamate or 50 μM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore but could be explained by anion permeation through independently functioning subunits. To further test the subunit independence, we coexpressed EAAC1WT and EAAC1H295K, a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1WT, and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1H295K and EAAC1WT, respectively. Finally, we coexpressed EAAC1WT with the pH-independent mutant transporter EAAC1E373Q, showing two independent populations of transporters, one being pH-dependent and the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits but that the individual subunits in the trimer function independently of each other.
Ultrasonic vibration controlled microfeeding is an accurate and precise method for dispensing and metering of fine powders. The signal amplitude of vibrational pulse in these micro-feeding devices is a key process variable affecting the precise and reproducible dosing of dry powders. The present research studies the effect of signal amplitude of an ultrasonic vibration controlled device on micro-dispensing of InhaLac®70, commonly used as a carrier in dry powder inhalations (DPIs). The experimental results show that a 0.4 – 1.6 mg mean dose mass range of InhaLac®70 could be accurately dispensed by varying the signal amplitude voltages from 2 – 5 V. Additionally, a linear increase in dispensed mean dose mass of InhaLac®70 could be noted with an increase in signal amplitude at a constant vibration pulse of 0.1 s and a nozzle diameter of 0.8 mm. High speed images captured during dispensing suggest that InhaLac®70 powder fell as discrete particles from the nozzle on application of ultrasonic vibration. The flow ceased as the vibration was stopped. These preliminary findings suggest that voltage amplitude of ultrasonic controlled micro-feeding device affects the micro-dispensing of mean dose mass of low dose, micron-sized InhaLac®70.
The applications of acoustic controlled micro-dosing system in accurate and precise dispensing of fine powders are well documented. Changes in the time period of ultrasonic vibration, a process variable in these devices affects the dosing process of fine powders. In the present work, the effect of different time period of vibration on the micro-dispensing behaviour of InhaLac®70, a carrier in dry powder inhalations (DPIs) is studied. The experimental results show that a wide and non-linear dose distribution of 1 – 10 mg of InhaLac®70 in a 0.8 mm nozzle and at fixed voltage signal amplitude of 5 V could be obtained on varying the time period of vibration in the range of 0.1 - 1 s. The dispensing of InhaLac®70 as discrete particles on initiation of ultrasonic vibration could be visualized using a high speed camera. The flow of particles ceased on stoppage of these vibrations. At the first 0.2s, the flow of InhaLac®70 particles was low, while it showed an increase above it. These changes suggested that different dosing processes could be modulated depending on time period of vibrations. These preliminary findings suggest that time period of vibrations in an acoustic micro-dosing system controls dispensed dosage of InhaLac®70.
Acoustic controlled micro-dosing is an accurate and promising method for dispensing and metering of fine powders. Ranges of nozzle sizes of these devices are needed for micro-dispensing of different powders, which in turn determines dispensed mean dose range. In the present work, selection of the nozzle size as a critical parameter in micro-dispensing of InhaLac®70, a carrier in dry powder inhalations (DPIs) is investigated. The experimental results show that mean dose mass range of 0.68 – 7.35 mg of InhaLac®70 could be accurately dispensed from nozzle size range of 0.6 – 0.9 mm. The high speed camera image of InhaLac®70 suggested that InhaLac®70 powder fell as discrete particles from the nozzle on initiation of ultrasonic vibration with flow of particles ceasing on stoppage of these vibrations. The arching behaviour of InhaLac®70 particles in smaller size nozzles and tending to compact and form lower strength arches in larger size nozzles lead to differences in dispensing of mean dose masses. These preliminary findings suggest that selection of nozzle size of acoustic controlled micro-feeding device is a key process parameter affecting the micro-dispensing of mean dose mass of low dose, micron-sized InhaLac®70.
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