SQI-Pr

SQI-Pr (Figure 8.2) is an electrogenic sodium (Na+) ionophore developed by Drs. Tsien and Minta. The SQI-Pr ionophore facilitates the transport of Na+ ions through lipid membranes, with the net effect of allowing Na+ to flow from a compartment with higher Na+ concentration, through the membrane, into a compartment with lower Na+ concentrations. These ionophores can be used to equilibrate intracellular and extracellular Na+ concentrations.

SQI-Et and SQI-Pr have an intrinsic fluorescence excitable by UV light. Therefore, they are not well-suited for use with SBFI, which is a UV-excitable Na+ indicator. These ionophores are ideally suited for use in conjunction with long-wavelength Na+ indicators like ANG-1 and ANG-2.

DATA

 

Emission: 475 nm

Effective Concentration:800-900 nM

Selectivity: 100:1 (Na+/K+)

Molecular Weight:  733 g/mol

RESULTS

 

SQI-Pr

Figure 8.2 SQI-Pr


SQI-Pr sodium Ionophore with ANG-2

Figure 8.5 Response of intracellular Asante Natrium Green 2 (ANG-2, Section 2.2 )to Na+ influx facilitated by SQI-Pr: REF52 fibroblasts loaded with ANG-2 (through incubation with AM ester) were maintained in Hanks’ Balanced Salt Solution (HBSS). SQI-Pr is a Na+ ionophore that is expected to promote Na+/H+ exchange across the cell membrane, and thus to cause Na+ influx. Upon application of 40 µM SQI-Pr, the fluorescence of intracellularly-loaded ANG-2 increased steadily, reflecting a rise in intracellular [Na+]. Further addition of 20 µM amphotericin B, a polyene natural product that increases membrane permeability to all the common monovalent ions (Na+, K+, H+ and Cl-), gave a small additional increment of indicator fluorescence, as expected. This experiment demonstrates the utility of ANG-2 as a sodium indicator, and the efficacy of SQI-Pr as a sodium ionophore. (Response shown is the average from 30 cells.)


SQI-Pr effect on membrane polorization

Figure 8.3: Effect of SQI-Pr on membrane polarization (Em). Neutrophils were equilibrated with 25 nM DiS-C3 in Na+-rich medium, and fluorescence was recorded.  Where indicated, 765 nM SQI-Pr was added. SQI-Pr promoted Na+ influx, which depolarized the membrane to positive voltages (towards the equilibrium potential of Na+), causing an increase in DiS-C3 fluorescence. Subsequent addition of 5.9 µM gramicidin D, an ionophore that transports both Na+ and K+, drove the membrane potential toward 0 mV, decreasing DiS-C3 fluorescence in the process.

SQI-pr

Figure 8.4: Increase in the fluorescence of membrane potential indicator DiS-C3 due to depolarization by SQI-Et (top) and SQI-Pr (bottom).  Red blood cells (RBC’s) were exposed to DiS-C3, and their membranes were subsequently depolarized by the SQI ionophores, resulting in an increase in DiS-C3’s fluorescence. Hyperpolarization caused by valinomycin is seen as a decrease in fluorescence.

 

 

Methods for Figure 8.5: REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM Asante Natrium Green 2 AM ester in bicarbonate-buffered Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, under an atmosphere of 5% CO2/95% O2; the medium also contained 0.0075%  (w/v) of the nonionic surfactant, Pluoronic F-127. The cells were then transferred into HBSS. SQI-Pr was prepared as a 20 mM stock solution in DMSO; amphotericin B was prepared as a 50 mM solution in DMSO. Appropriate volumes of the stock solutions were added to the HBSS bathing the cells to achieve the desired final concentrations used in the experiment. Indicator fluorescence was excited at 488 nm; fluorescence images were acquired with a cooled CCD camera.  [J Kao, Univ. of Maryland Medical School]

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