【正文】 Mark Sansom。 Conditions 100 Implications for Traditional Biochemistry Traditional Biochemistry (more or less) Ignores the Electric Field 101 But Rate Constants depend steeply on Concentration and Electrical Properties* because of shielding, a fundamental property of matter, independent of model, in my opinion. *nearly always 102 Electrostatic Contribution to ?Dissociation Constant? is large and is an Important Determinant of Biological Properties Change of Dissociation ?Constant? with concentration is large and is an Important Determinant of Biological Properties 103 Traditional Biochemistry ignores Shielding and Crowded Charge although Shielding Dominates Properties of Ionic Solutions and cannot be ignored in Channels and Proteins in my opinion 104 Make a Calcium Channel using techniques of molecular geics, sitedirected Mutagenesis How can we use these ideas? Gee Robillard, Henk Mediema, Wim Meijberg BioMaDe Corporation, Groningen, Netherlands 105 More? 106 Function can be predicted From Structure given Fundamental Physical Laws (sometimes, in some cases). 107 Strategy Use sitedirected mutagenesis to put in extra glutamates and create an EEEE locus in the selectivity filter of OmpF Sitedirected mutagenesis R132 R82 E42 E132 R42 A82 Wild type EAE mutant E117 E117 D113 D113 Gee Robillard, Henk Mediema, Wim Meijberg BioMaDe Corporation, Groningen, Netherlands 108 1 0 0 5 0 50 1 0 0 1 5 0 5 0501 5 0ECaWTE A EC u r r e n t (p A )V o l ta g e ( m V ) C i s T r a n s1 M C a C l2 0 .1 M C a C l2Ca2+Ca2+I VPL O TC i s T r a n sC i s T r a n sI V p l o t E A E : c u r r e n t r e v e r s e s a t e q u i l i b r i u m p o t e n ti a l o f C a2+ (ECa),i n d i c a t i n g th e c h a n n e l c a n d i s c r i m i n a t e b e t w e e n C a2+ a n d C lZerocurrent potential or reversal potential = measure of ion selectivity Henk Mediema Wim Meijberg 109 P Ca /P ClWT AAA 25EAE 100Ca2+ over Cl selectivity (PCa/PCl) recorded in 1 : M CaCl2 SUMMARY OF RESULTS (1) Conclusions: Taking positive charge out of the constriction zone (?= 3, see control mutant AAA) enhances the cation over anion permeability. Putting in extra negative charge (?= 5, see EAE mutant) further increases the cation selectivity. Henk Mediema Wim Meijberg 110 P Ca /P NaWT 2. 2AAA 3. 7EAE 4. 2Ca2+ over Na+ selectivity (PCa/PNa) recorded in M NaCl : M CaCl2 SUMMARY OF RESULTS (2) Conclusion: Compared to WT, EAE shows just a moderate increase of the Ca2+ over Na+ selectivity. To further enhance PCa/PNa may require additional negative charge and/or a change of the ?dielectric volume?. Work in Progress! Henk Mediema Wim Meijberg 111 Selectivity Differs in Different Types of Channels Wolfgang Nonner Dirk Gillespie Other Types of Channels 112 Ca channel Na channel Cl channel K channel prefers Small ions Ca2+ Na+ prefers Small ions Na+ Ca2+ Na+ over K+ prefers Large ions prefers K+ Na+ Selectivity filter EEEE 4 ? charges Selectivity filter DEKA 2 ?, 1+ charge Selectivity filter hydrophobic partial charges Selectivity filter single filing partial charges PNP/DFT Monte Carlo Bulk Approx Not modeled yet Selectivity of Different Channel Types The same crowded charge mechanism can explain all these different channel properties with surprisingly little extra physics. 113 Sodium Channel (with D. Boda, D. Busath, and D. Henderson) Related to Ca++ channel ? removing the positive lysine (K) from the DEKA locus makes calciumselective channel High Na+ selectivity ? 1 mM CaCl2 in M NaCl gives all Na+ current (pare to calcium channel) ? only 10 mM CaCl2 gives substantial Ca++ current Monte Carlo method is limited (so far) to a uniform dielectric Stay tuned…. Wolfgang Nonner Dirk Gillespie 114 Na+ Ca++ Na+/Ca2+ Competition in the Sodium Channel Biological Region Ca++ in bath (M) Wolfgang Nonner Dirk Gillespie 115 ?Model gives smallion selectivity. ?Result also applies to the calcium channel. Na+/Alkali Metal Competition in Na+ Channel Biological Region Wolfgang Nonner Dirk Gillespie 116 ?New? result from PNP/SPM bined analysis Spatial Nonuniformity in Na+ Channel Wolfgang Nonner Dirk Gillespie 117 Na+ vs K+ Selectivity Na+ K+ Channel Protein Protein Protein Protein Na+ Channel Wolfgang Nonner Dirk Gillespie 118 Na+ Channels Select Small Na+ over Big K+ because (we predict) Protein side chains are small allowing Small Na+ to Pack into Niches K+ is too big for the niches! Wolfgang Nonner Dirk Gillespie Summary of Na+ Channel 119 Sodium Channel Summary Na+ channel is a Poorly Selective Highly Conducting Calcium channel, which is Roughened so it prefers Small Na+ over big K+ Wolfgang Nonner Dirk Gillespie 120 Cl? Selective Channel Selective for Larger Anions The Dilute Channel Wolfgang Nonner Dirk Gillespie 121 Chloride Channel Channel prefers large anions in experiments, Low Density of Charge (several partial charges in nm3) Selectivity Filter contains hydrophobic groups ? these are modeled to (slightly) repel water ? this results in largeion selectivity Conducts only anions at low concentrations ? Conducts both anions and cations at high concentration Current depends on anion type and concentration Wolfgang Nonner Dirk Gillespie Doug H