Ion Channels: Proteins with a Hole

1. Chemist’s View IonChannelsProteins with a Hole All Atoms View Chemical Bonds are lines Surface is Electrical Potential Red is positive Blue is negative ~30 Å Figure by Raimund Dutzler

2. ION CHANNELS: Proteins with a Hole Channels form a class of Biological Systemsthat can be analyzed with Physics as Usual Physics-Mathematics-Engineering are the proper language for Ion Channels in my opinion

3. Ion Channels can be analyzed with Physics as Usual along with Biology as Usual to deal with the variety of channels of different structure and function

4. Biology is first of all a Descriptive Science Biology Involves Many Objects. Devices and Machines of Biology must be Identified and Named Before their Device Equation can be written. Just as engineers would name and describe devices! (Thousands of types of channels are essential for biological function)

5. Mathematics of Devices distinguishes Engineering from Physics (specifically spatially non-uniform boundary conditions), in my view. Unfortunately, this is not the time to justify this view! After they are named and described, by engineers or biologists, Devices can be understood by Physics as Usual Device Equations Specifying Output, given Input, are determined by the Physics of the Device and its Structure

6. Physics as Usual: Guess, Think and Check along with Biology as Usual a descriptive science: do everything “Why think? . . . Exhaustively experiment. Then, think” Claude Bernard Cited inThe Great Influenza, John M. Barry, Viking Penguin Group 2004

7. Biology as Usual: Cells and Channels Channels control flow in and out of cells ~5 µm Flow takes 0.1 msec to min

8. Ion Channels are the Main Molecular Controllers“Valves”ofBiological Function Flow takes 0.1 msec to min ~30 Å

9. Channels control flow of Charged Spheres Channels have Simple Invariant Structure on the biological time scale. Why can’t we predict the movement of Charged Spheres through a Hole? Physics of Ionic Movement under biological conditions is MUCH SIMPLER than the physics of Fluids, Plasmas, or Semiconductors, Fluids, plasmas, and semiconductors can be computed more or less perfectly. What is the Device Equation of a Channel? Ion Channels are Physical Devices

10. Ion Channels are Engineering Devices Channels Control Macroscopic Flow with Atomic Resolution

11. Ion channels respond to verysmall stimuli: chemicals, voltage, pH, and mechanical force.Gate/Switchfrom conducting to nonconducting state. Ion channels have Selectivity. Calcium channel selects Ca++ over Na+ by ~104. Ion channel proteins allowAtomic Scale Mutationsthat modify conductance, selectivity, and function. Ion channels are biologicalDevices that self-assemble into perfectly reproducible arrays. Ion channelsareTemplatesfor design of bio-devices and biosensors. Ion channelshaveVERY Large Charge Densities critical to I-V characteristics and selectivity (and gating?). ~30Å Ion Channels are Biological Devices Natural nano-valves* for atomic control of macroscopic flow Figure by Raimund Dutzler *nearly pico-valves: diameter is 49 Å

12. Ion Channels are Biological Devices Biological function of most channels is to allow andControl Flux of Ions Flux must follow a reasonably robust ‘law’ or the animal will die! Our task is to Discover, Understand, Control and Improve that law Thousands of scientists study channels every day ‘Law’ = Device Equation, not physical law

13. Ion Channelsare Important Enough to be Worth the Effort • Controller of most biological functions • Substantial fraction of all drugs work on channels • Simple enough to allow physical analysis of biological function • Thousands (literally) of papers at Biophysical Society Meeting Thousands of scientists study channels every day

14. Goal: PredictFunction From Structure given Fundamental Physical Laws Function is the current through the channel

15. Current in One Channel Molecule is a Random Telegraph Signal

16. Single Channel Currents have little variance John TangRush Medical Center

17. Single Channel Recording Patch clampand Bilayer apparatus clamp ion concentrations in the baths and thevoltageacross membranes. PatchClamp Setup Recordings from One Molecule

18. Single Channel Recording

19. OmpF KCl 1M 1M || G119D KCl 1M 1M || G119D KCl0.05 M 0.05M || ompF KCl0.05 M 0.05M || Current depends on Bilayer Setup Voltagein baths Concentrationin baths Fixed Chargeon channel protein John TangRush Medical Center

20. OmpF KCl 1M 1M || OmpF CaCl2 1M 1M || Current depends on type of ionSelectivity John TangRush Medical Center Bilayer Setup

21. Goal: Predict Function FromStructure given Fundamental Physical Laws

22. Connection to Molecular Biology Structures are inherited Genes control Structures Some structures are under partial experimental control Protein ‘folding’ Problem

23. Structures… Location of charges are known and can be controlled with atomic precision (~0.1 Å) in many favorable cases, e.g. porinby manipulating the genome

24. G119D Ompf Charge Mutation in Porin Structure determined by x-ray crystallography in Tilman Schirmer’s lab Figure by Raimund Dutzler

25. Goal: Predict Function From Structure given Fundamental Physical Laws

26. But … What are the Fundamental Physical ‘Laws’?

27. Verbal Models Are Popular with Biologists but Inadequate

28. James Clerk Maxwell “I only count molecules …., avoiding all personal enquiries which would only get me into trouble.” Slightly rephrased from Royal Society of London, 1879, Archives no. 188 in Maxwell on Heat and Statistical Mechanics, Garber, Brush and Everitt, 1995

29. I fear Biologists use Verbal Models which Maxwell avoided

30. Verbal Models areVagueandDifficult to Test Physicists avoid verbal models because …

31. Verbal Modelslead to Interminable Argument and Interminable Investigation Physicists avoid verbal models because …

32. thus,to Interminable Funding

33. and so Verbal Models Are Popular

34. Can Molecular Simulationsserve as “Fundamental Physical Laws”? Simulations are not Mathematics (e.g., results depend on numerical procedures and round-off error)

35. Can Molecular Simulationsserve as “Fundamental Physical Laws”? Only if they count correctly Simulations are Reliable Science when they are Calibrated

36. Simulations need validation and calibration as a new kind of science, in my opinion. Simulations are quite different from theory and experiment

37. First Principle of Numerical Integration The more work done, the larger the calculation, the more the error First Principle of Experimentation The more work done, the larger the effortthe less the error Simulations are not like experiments

38. Calibration… from truisms to specifics, It is very difficult for Molecular Dynamics to count well enough to reproduce ConservationLaws(e.g., of energy) Concentration (i.e., number density) or activity Energy of Electric Field Ohm’s ‘law’(in simple situations) Fick’s ‘law’(in simple situations) Fluctuationsin number density (e.g., entropy)

39. Can Molecular Simulations serve as “Fundamental Physical Laws”? What should be calibrated? I believe Thermodynamics of ions must be calibrated, i.e., activity = free energy per mole, which means the Pair Correlation Function according to classical Statistical Mechanics of Fluids

40. Calibrated Molecular Dynamics may be possible Pair Correlation Function in Bulk Solution • MD without Periodic Boundary Conditions─ HNC HyperNetted Chain Saraniti Lab, IIT: Aboud, Marreiro, Saraniti& Eisenberg

41. Calibrated MD may be possible,even in aGramicidin channel 16 Na+ single channel currents Molecular Dynamics without Periodic Boundary Conditions BioMOCA Simulations 235ns to 300ns, totaling 4.3 μs.Mean I = 3.85 pA, 24 Na+ crossings per 1 μs van der Straaten, Kathawala, Trellakis, Eisenberg & Ravaioli

42. Until Simulations are Calibrated, or mathematics of simulation is available, we take anEngineering Approach Hierarchy of Theories of Different Resolution

43. Engineering Approach Guess and Check! When the hierarchy is adequate, Computations (almost) equal experiments. Structural Engineering Circuit Design Airplane Design Computer Designare done by Physics-as-Usual, not molecular dynamicswith few experiments, little trial-and-error even though the physics is much more complex than electrodiffusion through holes

44. If engineering approach succeeds with biomolecules Benefits would be large to science, technology, and medicine. Experimental Effort needed without theory is Enormous! Significance of benefits is shown by the NIH 2004-5 budget = $2.8×1010 devoted almost entirely to descriptive experimental work

45. Experimental Effort needed without theory is Enormous Trial-and-Error Biology is very inefficient but it is (almost) All we have available, today.

46. Experimental Effort without theory is Enormous What is the alternative? Engineering Approach exploiting Physics of Biomolecules

47. Engineering Approach Essence of Engineering is knowing What Variables to Ignore! WC Randels quoted in Warner (2001) IEEE Trans Circuit Theory 48:2457

48. What variables should we ignore? when we make low resolution models. Use the scientific method Guess and Check! Intelligent Guesses are MUCH more efficient Sequence of un-intelligent guesses may not converge! (e.g., Rate/State theory of channels/proteins)

49. Guess Cleverly! Use Theory of Inverse Problems (Reverse Engineering) optimizes “Guess and Check” (1) Measure only what can be measured (e.g.,do not measure two resistors in parallel). (2) Make measurements that determine important parameters. (3) Use efficient estimators. (4) Use estimators with known bias These issues arise in any theory or simulation Heinz Engl, Martin Burger, Univ. of Linz, Austria

50. Channels are only Holes Why can’t we have a fully successful theory? Must know physical basis to make a good theory Where do we start?