Mediated transport processes are usually divided into two: passive facilitated diffusion and active transport. The latter is energy dependant, and is divided into primary and secondary types. Active transport can maintain large concentration differences between compartments, using metabolite energy. It takes place in the absence of passive forces and can be pharmacologically inhibited.
Active transport mechanisms are easily studied by the Ussing chamber (Fig. 1), an apparatus comprising two cells, separated by an epithelium of interest. A pair of voltage electrodes measures the potential difference between the two cells. Two other electrodes can apply a compensatory current, the so-called short-circuit current (Isc). Isc amplitude is selected so that the potential difference across the epithelium is set to zero. Ions can be traced in an Ussing chamber by radioactive labeling.
Fig.1: An Ussing chamber
A researcher filled an Ussing chamber with equal volumes of identical ionic solutions. After observing ion transport between the two cells, He used the following formula (Ussing equation), which is derived from the Nernst equation, to measure the relationships between ion fluxes, ion concentrations and voltage gradients:
In the formula, J12/J21 is the flux ratio (12 and 21 designate directions), C1/C2 is the concentration ratio on both membrane sides, (Ψ1-Ψ2) is the membrane potential, Z is the ion valence, F and R are constants and T is the temperature.
Questions:
1. According to the passage, which of the following is true? A) If the two cells of an Ussing chamber contain equal volumes of uncharged particles, the flux ratio will be concentration dependent.
B) If the two cells of an Ussing chamber contain the same volume and concentration of a non-ionic solution, a driving force can lead to a flux ratio of 1.
C) If the two cells of an Ussing chamber are filled with the same solution volume, a short circuit current has to be applied to maintain a voltage difference between the cells.
D) When the two cells comprising an Ussing chamber contain the same volume and the flux ratio is 1, a short circuit current cannot be applied to measure ion movement across the epithelium.
Uncharged particles cancel the electric gradient component, which might exist between the two sides of the epithelium. Since the volumes are also equal, only a concentration gradient can affect the flux ratio. Therefore, A is the correct answer. B is incorrect, since equal concentration and volume eliminate any possible driving force. C is incorrect, since equal cell volumes eliminate any possible hydrostatic force. However, it doesn't necessarily follow that a voltage gradient is established between the two cells. Therefore, Isc may not be required to maintain such a gradient. D is incorrect, because when the flux ratio is 1, a voltage difference can still exist between the two cells. This voltage difference will generate ion movement which can be balanced (and in that way also measured) by the short circuit current in the reverse direction.
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2. Active and passive transport mechanisms cannot be distinguished based on:
A) observing a linear curve for diffusion and a saturating curve for active transport, when plotting initial flux as a function of concentration difference in the same concentration range.
B) observing Michaelis-Menten kinetics and a gradient dependant flux for facilitated diffusion mediated by a transporter.
C) measuring a zero short circuit current when only passive forces drive transport.
D) noticing net change in pump activity when a putative inhibitor competes with an ion on binding.
Noticing net change in transporter activity is not sufficient for distinction, since both active carriers and channels can be affected by competitive inhibitors. Therefore, D is the correct answer. A is a good criterion for distinguishing passive from active transport, since in diffusion processes, the higher the gradient, the higher the flux. For active transport, there is saturation of initial flux rate, because of binding limitations. B is also a good criterion since facilitated diffusion acts in accordance with concentration gradients, and follows Michaelis-Menten kinetics, in contrast to active transport. C constitutes a good criterion as well, since according to the text, a zero short circuit current negates the option of passive transport and therefore can suggest that the transport is active thus distinguishing between the two modes of transport.
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