Dirk Gillespie Department of Molecular Biophysics and Physiology Rush University Medical Center

1. Dirk Gillespie Department of Molecular Biophysics and Physiology Rush University Medical Center Chicago, Illinois dirk_gillespie@rush.edu

2. Outline • review my model of ion current through RyR • current/voltage curves of single, open RyR • a new prediction of the model • RyR loses Ca2+ selectivity in high [divalent] • a different perspective on RyR selectivity • RyR is a Mg2+ channel • implications of Ca2+ and Mg2+ selectivity in disease • Mg2+ affects both Ca2+ current, as well as gating

3. Modeling RyR

4. What Do We Know About RyR Selectivity Filter? Gerhard Meissner’s lab • selectivity controlled by DDDD locus • D4899N • reduces conductance to 10-20% of normal • reduces PCa/PK from 7 to 1 • two other amino acids are also important • E4900Q and D4938N each reduce conductance to ~60% of normal and PCa/PK to 3 L. Gao et al., Biophys. J. 79, 828 (2000). Y. Wang et al., Biophys. J. 89, 256 (2005). L. Xu et al., Biophys. J. 90, 443 (2006).

5. cytosol SR lumen What Is in the Model • RyR pore • 5 Asp and Glu that affect permeation & selectivity • pore radius based on cryo-EM & experiments • ions • charged, hard spheres with crystal diameters • aspartate and glutamates • mobile oxygen atoms of terminal COO- groups • current • 1D Nernst-Planck D. Gillespie et al., J. Phys. Chem. B 109, 15598 (2005). D. Gillespie, Biophys. J. 94, 1169 (2008).

6. What Is in the Model • density functional theory (DFT) of ions • state-of-the-art in theory of inhomogeneous fluids • minimizes free energy of the system • electrolytes, colloids, polymers D. Gillespie et al., J. Phys. Chem. B 109, 15598 (2005). D. Gillespie, Biophys. J. 94, 1169 (2008).

7. Samsó et al., 2005 What is NOT in the Model • 99+% of this 2.3 MDa protein • only the pore near the selectivity filter is modeled • that section is modeled as 5 aspartates and glutamates

8. What is NOT in the Model • ion dehydration • in K+ channels, ions must shed waters before entering the very narrow selectivity filter • but, in RyR, this does not seem to be important • Mg2+ is equally as permeable as Ca2+ (PMg/PCa ~1) • why? • dehydration is actually a 2-step process: • shedding waters (an energetic penalty) • resolvation by the amino acids of the selectivity filter (an energetic advantage) • in RyR, this balance seems be 0 • if it’s important, the model won’t work

9. Comparisons to Experiments • with K+ only • WT – 19 ion conditions, +/- 150 mV • 5 mutations – 1 condition, +/- 150 mV • other monovalents • WT – 4 or 5 Li+, Na+, Rb+, and Cs+ conditions, +/- 150 mV • monovalent mixtures • bi-ionic – 7 conditions, +/- 150 mV • mole fraction – 6 conditions, +/- 150 mV • Ca2+ or Mg2+ and monovalent mixtures • WT –17 conditions (+/- 150 mV) & 11 at 0 mV • 3 mutations – 1 condition, +/- 150 mV • physiological – 1 condition, +/- 40 mV 87 uniqueion conditions

10. Predictions of Experiments • anomalous mole fraction effects (AMFEs) • Na+ + Cs+ – 5 conditions, +/- 150 mV • Li+/Na+/K+/Cs+ + Ca2+ – 4 conditions each, 0–30 mV • monovalent concentration dependence – 8 conditions • voltage dependence • monovalent mixtures • 3 species – 42 conditions, +/- 150 mV • “salting out” – 16 conditions, +/- 150 mV • reduction of Ca2+ selectivity at high divalent concentration • 6 conditions at 20 mV • physiological roles • RyR conducts majority of countercurrent during Ca2+ release • pathological changes in [Mg2+] affect Ca2+ release 91 uniqueion conditions

11. KCl cytosolic | lumenalconcentrations D. Gillespie, Biophys. J. 94, 1169 (2008).

12. KCl 250 mM cytosolic | x (25-2000 mM) lumenal concentrations

13. CsCl CaCl2 NaCl CaCl2 Divalent Cations 250 mM | 250 mM monovalent 4 μM | ■ 5 mM divalent ■ 10 mM ■ 50 mM bi-ionic: ▲250 mM monovalent | 25 mM divalent D. Gillespie, Biophys. J. 94, 1169 (2008).

14. Mole Fraction Experiments • The model predicted an AMFE when Ca2+ is added to 100 mM Na+ or Cs+ with different • depths • effects of low [Ca2+]lum D. Gillespie, Biophys. J. 94, 1169 (2008).

15. RyR selectivity

16. screening advantage number advantage mean electrostatic advantage excluded-volume advantage Energetics of Selectivity • Each term of the electrochemical potential contributes to the ratio of ion concentrations in the pore: D. Gillespie, Biophys. J. 94, 1169 (2008).

17. Energetics of Selectivity • 150 mM KCladd CaCl2 • mean-field advantage disappears • Ca2+ maintains screening advantage • excluded volume insignificant favors Ca2+ binding favors K+ binding RyR is a calcium channel because Ca2+ is coordinated (screened)by the aspartates better than K+. D. Gillespie, Biophys. J. 94, 1169 (2008).

18. Model Prediction • As [Ca2+] increases, Ca2+ vs. K+ selectivity decreases. • Moreover, this a divalent (not ionic strength effect). • Experiments: • measure current in symmetric [K+] (no Ca2+) • I0Ca • measure current with 100:1 [K+] to [Ca2+] ratio (luminal) • I+Ca • Plot fractional reduction of current (FRC) • low FRC means low Ca2+ selectivity • high FRC means high selectivity

19. RyR is a Mg2+ channel

20. Physiological Ionic Conditions • Under normal (resting) physiological conditions, D. Gillespie et al., Cell Calcium 51, 427 (2012).

21. Mg2+ Is the Most Abundant Ion • Because Mg2+ is present on both sides of RyR, it is the most abundant ion in the selectivity filter under physiological conditions. Over the physiological range of [Ca2+]SR and [Mg2+], the number of K+ in the filter changes little. Ca2+ and Mg2+ compete with each other for the pore. 1 mM Mg2+ Mg2+ Mg2+ 1 mM SR Ca2+ Ca2+ K+ K+ Ca2+ D. Gillespie et al., Cell Calcium 51, 427 (2012).

22. Ca2+ current in pathological conditions

23. Physiological Ionic Conditions • Under normal (resting) physiological conditions, • Under pathological conditions, cardiac [Ca2+] and [Mg2+] can increase/decrease by a factor 2. • it is mainly Ca2+ and Mg2+ that compete for the pore • this change in ion competition changes iCa D. Gillespie et al., Cell Calcium 51, 427 (2012).

24. Ca2+ Current Changes • Therefore any condition that changes [Ca2+]SR and [Mg2+], will make large changes in unitary Ca2+ current. This is in addition to the effect of Mg2+ on gating, which is of comparable size. D. Gillespie et al., Cell Calcium 51, 427 (2012).

25. Ca2+ Current Changes • In the worst cases, there are large changes in Ca2+ current that should have a significant effect on Ca2+ and therefore muscle function. D. Gillespie et al., Cell Calcium 51, 427 (2012).