Question

Pu010 2. You are studying a bacterial protein, Pmp 7, that pumps one potassium (Kt) ion into the cell for each calcium ion (Ca²+) that is moved out of the cell. Pmp 7 uses energy from light to pump these ions across the membrane. Experiment 1: You make an artificial membrane vesicle that has Pmp7 as the only protein on the membrane. Pmp 7 is orientated on this artificial membrane vesicle as it is oriented in a bacterial cell, and works just like it would in the cell. At the beginning of your experiment, the concentration of K+ and Ca2+ are at 100 uM both inside and outside of the vesicle. i) If you kept these vesicles in the dark, would you have a concentration gradient of K+ across the membrane of the vesicle? (2 points) ii) Now you take these vesicles and shine light on the vesicle. 1 minute after you shine light, would you predict the concentration of Ca2+ will be higher or lower inside the vesicle compared to the outside of the vesicle? (2 points) iii) Would you set up a membrane potential across the membrane under the conditions described in 2ii? Explain. (4 points) Experiment 2: You now make artificial membrane vesicles that have both the K+ leak channel and also Pmp7, oriented as it would be found on a bacterial cell. Remember that leak channels are not gated, but will open randomly. At the beginning of this experiment, the concentration of K and Ca2+ are at 100 um both inside and outside of the vesicle. iv) If you keep these vesicles (described for experiment 2) in the dark, would you set up a membrane potential across the membrane? Explain. (4 points) v) Now you take these vesicles from experiment 2 and shine light on them. 1 minute after you shine light, would you predict the concentration of Ca2+ will be higher or lower inside or outside the vesicle? (2 points) vi) For the vesicles from experiment 2 that are in the light (Q2v), which ion do you predict will be closest to its equilibrium potential? Explain. (4 points)

Answer 1

Answer of experiment no. 1:

i) Pmp7 transmembrane protein uses energy from light to pump ions in and out of the cell across the membranes. If the vesicles are kept in dark, there will not transport of ions and there will be no concentration gradient of K+ across the membrane of the vesicle.

ii) If the vesicles are exposed to light of one minute duration, there will influx of K+ ions into the cell and efflux of Ca2+ ions outside of the cell. Hence, the concentration of Ca2+ lower inside the vesicle compared to the outside of the vesicle.

iii) A membrane potential can be set up across the membrane under the conditions described in 2ii. Since there is influx and efflux of divalent (Ca2+) and monovalent (K+) ions across the membrane, there will generation of differential charges.   

    Answer of experiment no. 2:

i) There is a K+ leak channel in the artificial membrane vesicle in addition to the Pmp7. The leak channels are not gated and open randomly. If these vesicles are kept in the dark, a membrane potential across the membrane can be set up. Although the Pmp7 protein will not pump either the Ca2+ or K+, the K+ will enter into the cell through the leak channel.

ii) If the vesicles are exposed to one minute of shine light, the concentration of the Ca2+ will be lower inside the vesicle compared to the outside of the vesicle. The light will activate the pumping by Pmp7.

iii) For the vesicles that are in light, the ion that will be closest to its equilibrium potential is Ca2+. The K+ influx into the cell is not stable and properly regulated because of the extra K+ leak channel addition to the pump of K+ ions by   Pmp7 into the cell. The influx of K+ ions into the cell through the leak channel is random.