TestQuestions-Transport

Molecular Biochemistry I

Membrane Transport

Reading:

Textbook Reading: Biochemistry, 3rd Edition, by Voet & Voet, Chapter 20.

Some recent articles (optional reading):

J. Abramson, S. Iwata & H. R. Kaback (2004) "Lactose permease as a paradigm for membrane transport proteins," Molec. Membr. Biol. 21: 227-236.
L. Guan & H. R. Kaback (2006) "Lessons from Lactose Permease," Annu. Rev. Biophys. Biomol. Struct. 35: 67-91.
C. Xu, W. J. Rice, W. He & D. L. Stokes (2002) "A structural model for the catalytic cycle of Ca²⁺-ATPase," J. Mol. Biol. 316: 201-211.
D. L. Stokes & N. M. Green (2003) "Structure and function of the calcium pump," Annu. Rev. Biophys. Biomol. Struct. 32: 445-468.
C. Toyoshima & G. Inesi (2004) "Structural basis of ion pumping by Ca²⁺-ATPase of the sarcoplasmic reticulum," Annu. Rev. Biochem. 73: 269-292.
L. J. DeFelice (2004) "Transporter structure and mechanism," Trends in Neurosci. 27: 352-359.
H. S. Young & D. L. Stokes (2004) "The mechanics of calcium transport," J. Membr. Biol. 198: 55-63.
M. T. Facciotti, S. Rouhani-Manshadi & R. M. Glaeser (2004) "Energy transduction in transmembrane ion pumps," Trends in Biochem. Sci. 29: 445-451.
K. Obara, N. Miyashita, C. Xu, I. Toyoshima, Y. Sugita, G. Inesi & C. Toyoshima (2005) "Structural role of countertransport revealed in Ca²⁺ pump crystal structure in the absence of Ca²⁺," PNAS 102: 14489-14496.
T. L.-M. Sorensen, C. Olesen, A.-M. L. Jensen, J. V. Moller & P. Nissen (2006) "Crystals of sarcoplasmic reticulum Ca²⁺-ATPase," J. Biotech. 124: 704-716.
M. Cahalan & E. Neher (1992) "Patch clamp techniques: An overview," Methods in Enzymology 207: 3-14 (and accompanying articles).
K. Jurkat-Rott & F. Lehmann-Horn (2004) "The patch clamp technique in ion channel research," Curr. Pharmaceut. Biotech. 5: 387-395.

Potential Test Questions:

1. Describe the structure and proposed mechanism of action of the ionophore valinomycin. How is the structure of valinomycin suited to its role? Contrast valinomycin with the structure of the lactose permease. How does the mechanism of solute translocation for a carrier protein like lactose permease differ from that of valinomycin?

2. Diagram and describe the structure of the gramicidin channel. Explain the fluctuations in current observed when a voltage is applied across a membrane containing gramicidin, bathed on both sides with salt solution. What is thought to be the mechanism of gramicidin gating (channel opening and closing)?

3. Describe the Ca⁺⁺-ATPase (SERCA) of endoplasmic reticulum membranes, including its reaction cycle, its functional activity, and its homology to other known ion pumps. On the diagram at right, indicate the approximate location of each of the following domains, and indicate their roles: membrane domain, cytosolic domain, Ca⁺⁺-binding sites, Asp351. What is the role of Asp351?

4. a. What is the basis for the fluctuations in recorded current in a patch clamp recording from a membrane containing a single ion channel? Diagram and explain how channel conductance is determined from such a patch clamp recording.
b. How can current amplitude histograms (summarizing occupancy of different current levels during patch clamp recordings) be used to test for factors that regulate channel gating? What would be the expected effect on the current amplitude histogram of a reagent that induces channel opening?

Studio Exercise: Patch Clamping Data Analysis

A brief recording of current from an excised patch containing apparently a single ion channel is shown below. Current was recorded with the voltage clamped at -60 mV, in medium of 150 mM K⁺. Each line of recording represents 102 milliseconds.

In the diagram below the bar at left is a scale marker designating 20 pico-Amperes of current (20 X 10^-12 Amp). Note that at a negative voltage, channel opening results in a downward deflection, corresponding to a larger negative current (see at right).

The current amplitude histogram at right was generated with the program PCLAMP. It summarizes the occupancy of current levels during a few seconds of the same recording. The vertical axis is a compilation of the amount of time the channel spent at each current level.

If the patch contains a single channel that fluctuates between 2 states, open and closed, there should be 2 peaks in the current amplitude histogram, representing open and closed states. The baseline current, when the channel is closed, is due to leakiness of the seal between the membrane and the patch pipet, and permeability of the bilayer membrane.

Recall that, according to Ohm's law, resistance (units of ohms) equals voltage divided by current, while conductance (the reciprocal of resistance, designated in units of Siemens) equals current divided by voltage.

Problem and Questions for in-class discussion:

Using a ruler and the scale bar on the current recording, estimate the average current increment, in picoAmperes (10^-12 Amp), with channel opening. Then estimate the single channel conductance (reciprocal of resistance) in pS (10^-12 Siemens), using the relationship: conductance = current/voltage, or Siemens = Amperes/Volts. Current flow through the open channel would vary with voltage, which is the driving force for transmembrane ion flux. The single channel conductance, which is usually independent of voltage, is characteristic of a given channel type.
Identify the peaks in the current amplitude histogram corresponding to open and closed states. Estimate the average current transition from the amplitude histogram, and compute the single channel conductance from that value.
Did the channel spend more time open or closed during the time period summarized in the current amplitude histogram? How could the percentage "open probability" be estimated from the amplitude histogram?
Compare what would you expect to see in current amplitude histograms summarizing recordings in the presence and absence of a ligand that induces channel opening. Explain.

Lecture notes on
Membrane Transport

Interactive Quiz on
Membrane Transport