Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching

Characterizing membrane proteins by sedimentation equilibrium is challenging because detergents and/or lipid molecules, usually required for solubilization, form a complex with the protein. The most common way to overcome this problem is Tanford and Reynolds' density matching method, which eliminates the buoyant mass contributions of detergents/lipids by adjusting the solvent density with D2O/H2O mixtures to render either detergent or lipid molecules neutrally buoyant. Unfortunately, the method is practical only for detergent densities between 1.0 (H2O) and 1.1 (D2O) g ml(-1), excluding many of the more commonly used detergents for membrane protein studies. Here, we present a modern variant of Tanford and Reynolds' method that (1) is applicable to any detergent regardless of its specific density, (2) does not compromise accuracy and precision, and (3) provides additional information about the number of detergent molecules that are bound to each protein. The new method was applied successfully to Delta(1-43)A-I, an amino-terminal deletion mutant of human apolipoprotein A-I. Interestingly, we observed a significantly lower Delta(1-43)A-I/octyl-glucoside complex partial specific volume than that expected from volume additivity rules, indicative of specific protein-detergent interactions.     

Investigation of the electron transfer reactions and redox characteristics of photoactive bacteriochlorophyll in Rhodobacter sphaeroides reaction centers modified by D2O and cryoprotectants

The effects of D2O, glycerol and dimethyl sulfoxide (DMSO) on redox potential Em of bacteriochlorophyll of a special P2 or [P(M)P(L)] pair, the rate of energy migration from bacteriopheophytin H(M) to [P(M)P(L)], electron transfer from [P(M)P(L)] to bacteriopheophytin H(L) and then to quinone Q(A) in reaction centers (RC) of Rhodobacter sphaeroides were studied. The H2O --> D2O substitution did not change Em of the special pair, whereas addition of 70% glycerol or 35% DMSO (v/v) increased the values of Em by 30 and 45 mV, respectively. Rate constants of energy migration km(H(M)* (km)--> P2), charge separation ke([P(M)P(L)] *H(L) (ke)--> [P(M)P(L)] +H(L)-), electron transfer to quinone kQ did not change after the glycerol addition, whereas isotopic substitution and addition of DMSO caused a 2-3-fold increase in km, ke, and kQ values. Theoretical analysis of the redox center potential dependence on dielectric permeability epsilon, swelling of the protein globule in a solvent, and on changes in the charge distribution (charge shifts) in the protein interior near the redox center was carried out. It has been shown that the H2O replacement with DMSO can result in the Em increase by tens of mV. No correlation was found between the Em values and the rate of charge separation upon isotopic substitution and addition of cryoprotectants. The effect of epsilon of the medium on the rate of electron transport due to changes of energy of intermolecular interaction between the donor and acceptor molecules was estimated.     

Solubility and phase transitions in the water-protein-salt system

The previously derived formulas for the curves corresponding to sol-gel, liquid-liquid, and liquid-solid phase transitions, which correlate the critical molar composition of the water-protein-salt system with individual characteristic features of its component (protein charge z, the number of ions adsorbed v, the function of electrolyte activity A) are presented as curves in ordinary coordinates of protein solubility logS against salt concentration m3. Tendencies in changes in phase transition lines versus the v, z, and v/z ratio have been determined. Correlations of the salting-out curve and the salting-out coefficient with phase transitions are discussed.     

Cell Wall Turnover at the Hemispherical Caps of Bacillus subtilis

Cell walls made by Bacillus subtilis bacteria grown in D(2)O medium have buoyant densities in CsCl which are different from walls made by cells grown in H(2)O medium. Cell wall turnover was studied by measuring the change in wall buoyant density after a B. subtilis culture was shifted from growth in D(2)O medium to aeration in H(2)O medium. Walls from the hemispherical caps were isolated after preferential digestion of wall from the cylindrical regions using the B. subtilis autolytic amidase. The walls from the polar regions were found to turn over extensively.     

Estimate of the Molecular Weight of the Sarcolemmal Sodium Channel Using H2O-D2O Centrifugation

The sodium channel saxitoxin binding component from rat sarcolemma was solubilized with NP-40 and centrifuged on sucrose gradients constructed in either D2O or H2O. When compared with a series of standard proteins the sedimentation behavior of the solubilized channel complex changed from an apparent S20,w of 9.1 in H2O to 6.1 in D2O. From these observations, a true partial specific volume of 0.83 ml/g was calculated for the complex. A Stokes radius of 8.6 nm was estimated from Sepharose 6-B chromatography in NP-40. The calculated protein molecular weight of the lipid-protein-detergent complex based on these data is 560,000. The complex contains about 56% protein, and the calculated molecular weight of this component is 314,000 if a v for the protein of 0.74 ml/g is assumed.     

Ternary copper(II) complexes with N-carboxymethyl-l-prolinato(2-) ion and imidazole or creatinine: a comparative study of the interligand interactions influencing the molecular recognition and stability

The compounds {[Cu(CMP)(Him)].H(2)O}(n) (I) and [Cu(CMP)(crea)H(2)O].3H(2)O (II) were synthesized and characterized by X-ray diffraction, thermal, spectral and magnetic methods (CMP=N-carboxymethyl-;l-prolinato(2-) ion, Him=imidazole and crea=creatinine). Appropriate structural comparison with other compounds such as {[Cu(CMP)(H(2)O)].H(2)O}(n), [Cu(crea)(2)Cl(2)] and [Cu(dipeptide)(crea)(H(2)O)(x)].nH(2)O (x=0 or 1) have been made in order to prove that crea can act as an imidazole-like ligand (because it is able to promote the same fac- to mer-CMP tridentate conformational change in copper(II) complexes) as well as to discuss the interligand interactions which control the 'Cu(CMP) complex-crea, molecular recognition processes. In contrast to that found in related ternary complexes, we have concluded that direct CMP-crea interligand interactions are missing in the Cu-CMP-crea complex due to the inappropriate correspondence between the donor and/or acceptor H-bonding properties of these ligands. CMP can only act as H-acceptor by its two terminal carboxylate group, and crea can display H-donor and H-acceptor roles by its exocyclic -NH(2) and O moieties, respectively. That promotes the reinforcement of the Cu-N(crea) bond by a bridge -N-H(crea)...O(aqua) (2.867(3)A, 176.4 degrees).     

Size characteristics of the solubilized saxitoxin receptor of the voltage-sensitive sodium channel from rat brain

The saxitoxin receptor of the voltage-sensitive sodium channel from rat brain was solubilized with Triton X-100 and stabilized with phosphatidylcholine. The size characteristics of the detergent . phospholipid . receptor complex were studied by gel filtration and sucrose gradient sedimentation in H2O and D2O. The complex has Stokes radius = 80 A, S20,W = 12 S, v = 0.82 ml/ g, and Mr = 601,000 +/- 48,000. Assuming v = 0.73 ml/g for the saxitoxin receptor protein, the mass of the complex consists of 47.4% detergent and phosphatidylcholine and 52.6% saxitoxin receptor protein with Mr = 316,000 +/- 63,000.     

Methanol-induced conformations of myoglobin at pH 4.0

The equilibrium methanol-induced conformation changes of holomyoglobin (hMb) at pH 4.0 have been studied by circular dichroism, tryptophan fluorescence, and Soret band absorption and by electrospray ionization mass spectrometry (ESI-MS). Optical spectra show the following: (1) In 35-40% (v/v) methanol/water, the native-like secondary structure remains, the tertiary structure is lost, the heme protein interactions are decreased, and a folding intermediate is formed. (2) In 50% methanol, heme is lost from the protein, and there is a small decrease in helicity together with a loss of tertiary structure. (3) At >60% methanol, the helicity increases and the apoprotein goes into a helical denatured state. The conformations are also probed by the charge states produced in ESI-MS and by hydrogen/deuterium (H/D) exchange with mass measurement by ESI-MS. At 0-30% methanol, native hMb produces relatively low charge states (9(+)-13(+)) in ESI-MS and exchanges relatively few hydrogens. In 35-40% methanol, at which an intermediate is formed, there is a bimodal distribution of hMb ions with both low (9(+)-13(+)) and high (14(+)-23(+)) charge states and also a high charge state distribution (12(+)-26(+)) of apomyoglobin (aMb) ions. Low and high charge states of hMb and a high charge state of aMb all show the same H/D exchange rate, indicating that an unfolded hMb intermediate interconverts between folded hMb and unfolded aMb. The charge state distribution for the unfolded hMb intermediate observed here is similar to that of the recently reported transient intermediate formed during the acid denaturation of hMb. At 50% alcohol the protein produces predominantly high charge states of aMb ions and shows H/D exchange rates close to those of the acid-denatured protein. H/D exchange of the helical denatured protein at alcohol concentrations >60%, at which high charge states of aMb are produced, shows that the protein structure is more protected than at approximately 50% methanol.     

Relation between structural-dynamic organization of reaction centers in Rhodobacter sphaeroides and picosecond steps of photosynthesis

The effect of deuteration, and the addition of glycerol and dimethylsulfoxide on the redox midpoint potential Em of bacteriochlorophyll of the special pair ?PMPL?, the rate of energy migration from bacteriopheophytin HM to ?PMPL?, and electron transfer from ?PMPL? to HL and from HL to quinone QA in reaction centers of Rhodobacter sphaeroides was studied. It was shown that H2O-->D2O substitution did not change Em of the special pair, while the addition of 70% glycerol and 35% dimethylsulfoxide (v/v) increased the Em value by 30 and 45 mV, correspondingly. The rate constants of energy migration [formula: see text], charge separation [formula: see text], electron transfer to QA kQ remained unchanged upon the addition of glycerol. The isotopic substitution of water and addition of dimethylsulfoxide led to a 2-3-fold increase in km, ke and kQ values. The dependence of the potential of redox center on the dielectric constant epsilon was analyzed. It was shown that replacement of H2O by dimethylsulfoxide can increase Em by tens of millivolt. There was no correlation between changes in Em and the values of km, ke and kQ upon deuteration and addition of cryoprotectors. It was concluded that the processes of energy migration, charge separation, and electron transfer to the quinone acceptor are preceded by the solvation of states H*M, ?P+MP-L?* and [formula: see text].     

Simultaneous determination of triazolam and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of triazolam and its metabolites, alpha-hydroxytriazolam (alpha-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), was developed and validated. Triazolam-D4 was used as the internal standard (IS). This analysis was carried out on a Thermo((R)) C(18) column and the mobile phase was composed of acetonitrile:H(2)O:formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) and quantification was performed by multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 343.1-->308.3, 359.0-->308.3, 359.0-->111.2 and 347.0-->312.0 for triazolam, alpha-OHTRZ, 4-OHTRZ and triazolam-D4, respectively. LLOQ of the analytical method was 0.05ng/mL for triazolam and 0.1ng/mL for alpha-OHTRZ and 4-OHTRZ. The within- and between-run precisions were less than 15.26% and accuracy was -8.08% to 13.33%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of triazolam in healthy Chinese volunteers.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation Ii. Physical Characteristics of Thymocyte Progenitor Cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H × AKR)F₁ bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, thymocyte progenitor cells show the same distribution as CFUs. the relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Adair was right in his time

The subunit molar mass of hemoglobin was established in the 19th century by chemical analysis, the tetramer structure by osmotic pressure determination in 1924 and by the newly developed analytical ultracentrifuge in 1926, which became a powerful tool for biological macromolecule molar mass determinations. The Svedberg equation was derived by eliminating the translational friction coefficient relating to sedimentation and diffusion in the ultracentrifuge in a strictly solute/solvent vanishing concentration two-component system analysis. A differential equation describing the radial equilibrium concentration distribution in the ultracentrifuge was also derived, both yielding the buoyant molar mass (1-nu2rho)M2 term. Many years later it was realized that solutions of biological macromolecules are multicomponent systems and the two-component analysis leads to minor or major erroneous results. Thermodynamic derivation of an equation for multicomponent systems redefines the buoyant molar mass terms by (deltarho/deltac2)muM2, leading to correct molar mass (g/mol) values following determination of the density increment at constant chemical potentials of diffusible solutes, and powerfully connects the analytical sedimentation equation to the osmotic pressure concentration derivative and, in a broad complementary sense, to light, X-ray and neutron scattering experiments. Macromolecular interactions can be studied with high precision and solute-solvent interactions yield powerful information relating to "thermodynamic" hydration, closely related to hydration derived from X-ray diffraction, as well as solute-cosolute interactions. A series of examples is given to demonstrate the correctness and usefulness of the thermodynamic multicomponent system approach. It is a strange fact that in current analytical ultracentrifugation analysis the elegant and powerful multicomponent solution technology is almost totally disregarded and the classical limited validity Svedberg approach is used uniquely.     

Molecular weight of detergent-solubilized prostaglandin-F2 alpha receptor from bovine corpora lutea

A prostaglandin F2 alpha receptor localized in plasma membranes of bovine corpus luteum cells was solubilized by treatment with Triton X-100. Sepharose chromatographies of ([3H]prostaglandin F2 alpha)-receptor complex gave a Stokes' radius of 630 nm. In the absence of detergent, aggregated forms of the receptor appeared. Sedimentation experiments of solubilized receptor in sucrose/H2O and sucrose/2H2O density gradients gave the following values: sedimentation coefficient (S20, w) 4.6 S; partial specific volume (VB) 0.78 cm3/g and frictional ratio (f/fo) 1.6. Based on the sedimentation coefficient and the Stokes' radius and assuming that the receptor is a non-glycosylated protein the molar mass of the receptor-(Triton X-100) complex was 144000 g/mol. The VB value indicated that ca. 26% of the weight represented bound detergent and that the molecular weight of the prostaglandin F2 alpha receptor is approximately 107000.     

Integrity of intracellular domain of Notch ligand is indispensable for cleavage required for release of the Notch2 intracellular domain

The biological activity of the soluble form of the Notch ligand (sNL) and requirement of the intracellular domain (ICD) of the Notch ligand have been debated. Here we show that soluble Delta1 (sD1) activates Notch2 (N2), but much more weakly than full-length Delta1 (fD1). Furthermore, tracing the N2 molecule after sD1 stimulation revealed that sD1 has a defect in the cleavage releasing ICD of N2 (intracellular cleavage), although it triggers cleavage in the extracellular domain of N2. This represents the molecular basis of the lower activity of sD1 and suggests the presence of an unknown mechanism regulating activation of the intracellular cleavage. The fact that Delta1 lacking its ICD (D1Delta(ICD)) exhibits the phenotype similar to that exhibited by sD1 indicates that the ICD of D1 (D1(ICD)) is involved in such an as yet unknown mechanism. Furthermore, the findings that D1Delta(ICD) acts in a dominant-negative fashion against fD1 and that the signal-transducing activity of sD1 is enhanced by antibody-mediated cross-linking suggest that the multi merization of Delta1 mediated by D1(ICD) may be required for activation of the N2 intracellular cleavage.     

Determination of tamsulosin in dog plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometric method is described for the determination of tamsulosin in dog plasma. Tamsulosin was extracted from plasma using a mixture of hexane-ethyl acetate (2:1, v/v) and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of methanol, water and formic acid (80:20:1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Atmospheric pressure chemical ionization (APCI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 409-->m/z 228 and m/z 256-->m/z 166.9 were used to quantify tamsulosin and the internal standard, respectively. The linearity was obtained over the concentration range of 0.1-50.0 ng/ml for tamsulosin and the lower limit of quantitation was 0.1 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 5.0 and 4.0% (relative standard deviation (R.S.D.)), respectively, and accuracy was within +/-0.3% (relative error (R.E.)). This method was successfully applied to pharmacokinetic study of a tamsulosin formulation product after oral administration to beagle dogs.     

Lateral diffusion of PEG-Lipid in magnetically aligned bicelles measured using stimulated echo pulsed field gradient 1H NMR

Lateral diffusion measurements of PEG-lipid incorporated into magnetically aligned bicelles are demonstrated using stimulated echo (STE) pulsed field gradient (PFG) proton (1H) nuclear magnetic resonance (NMR) spectroscopy. Bicelles were composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) (q = DMPC/DHPC molar ratio = 4.5) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000) at 25 wt % lipid. 1H NMR STE spectra of perpendicular aligned bicelles contained only resonances assigned to residual HDO and to overlapping contributions from a DMPE-PEG 2000 ethoxy headgroup plus DHPC choline methyl protons. Decay of the latter's STE intensity in the STE PFG 1H NMR experiment (g(z) = 244 G cm(-1)) yielded a DMPE-PEG 2000 (1 mol %, 35 degrees C) lateral diffusion coefficient D = 1.35 x 10(-11) m2 s(-1). Hence, below the "mushroom-to-brush" transition, DMPE-PEG 2000 lateral diffusion is dictated by its DMPE hydrophobic anchor. D was independent of the diffusion time, indicating unrestricted lateral diffusion over root mean-square diffusion distances of microns, supporting the "perforated lamellae" model of bicelle structure under these conditions. Overall, the results demonstrate the feasibility of lateral diffusion measurements in magnetically aligned bicelles using the STE PFG NMR technique.     

ESI-MS investigation of solvent effects on the chiral recognition capacity of tartar emetic towards neutral side-chain amino acids

The effect of solvent systems on previously-reported ESI-MS based proton-assisted enantioselective molecular recognition phenomena of tartar emetic, L-antimony(III)-tartrate, was evaluated. This was achieved by carrying out a series of competitive binding experiments using chiral selectors, bis(sodium) D- and -L-antimony(III)-tartrates with chiral selectands, neutral side-chain amino acid enantiomeric isotopomers of alanine (Ala), valine (Val), leucine (Leu) and phenylalanine (Phe), in three different solvent systems, ACN/H(2)O (75/25 v/v), H(2)O (100%) and H(2)O/MeOH (25/75 v/v). Observations from these experiments suggest that the effect of solvent systems on previously reported proton-assisted chiral recognition capacity of D,L-antimony(III)-tartrates is small, but not negligible. It was observed that an ACN/H(2)O (75/25 v/v) solvent system facilitates and enhances the chiral discrimination capacity of protonated {[D,L-Sb(2)-tar(2)][H]}(-) ionic species. Further, amino acid enantiomers showed a general trend of increasing selectivity order, Val ≤ Ala < Leu ≈ Phe towards the protonated {[D,L-Sb(2)-tar(2)][H]}(-) ionic species which was independent of the solvent system employed. The lack of enantioselective binding for {[D,L-Sb(2)-tar(2)]}(2-) ionic species was consistently recorded in respective mass spectra from all performed experiments, which suggests that ESI-friendly solvent systems have no effect and do not influence this phenomenon.     

Action of myeloperoxidase-hydrogen peroxide-chloride system on the egg white lysozyme

The enzyme system composed of human neutrophilic myeloperoxidase (H2O2-oxidoreductase, EC 1.11.1.7), H2O2 and Cl-, at pH 4.5 interacts with egg white lysozyme (EC 3.2.1.17) in several stages. In the first stage, occurring at lysozyme to H2O2 molar ratio of 1:1.4-1.8, the lysozyme loses its enzyme activity but does not yield any derivative distinguishable from the native protein on polyacrylamide gel electrophoresis (PAGE). The second stage of oxidation begins at lysozyme to H2O2 molar ratio above 1:5, producing a change in the lysozyme spectrum at 260-290 nm, and yielding protein derivatives with molecular masses equal to multiples of 14.3 kDa, i.e. the lysozyme molecular mass. This implies that an excessive oxidation of lysozyme by the myeloperoxidase-H2O2-Cl- system produces cross-linking of lysozyme molecules to di-, tri-, tetra-, and pentameric structures. At lysozyme to H2O2 molar ratio exceeding 1:12 a water insoluble white product, which consists of a set of lysozyme cross-linked derivatives, is obtained.     

Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia coli. Purification by high performance liquid chromatography and direct analysis by plasma desorption mass spectrometry

Lipopolysaccharide (LPS) extracted from the deep rough mutant of Escherichia coli D31m4 was disaggregated with 0.1 M EDTA, pH 7.0, and fractionated on a diethylaminoethyl-cellulose column to yield the biphosphate form of LPS. After methylation, the derivative was purified by reverse-phase high performance liquid chromatography using a C18-bonded silica cartridge. A linear gradient of 50-100% isopropyl alcohol/water (93:7, v/v) in acetonitrile/water (93:7, v/v) was used over a period of 60 min. The derivatized LPS showed a single major peak by high performance liquid chromatography, and this hexamethyl hexaacyl LPS was recovered and subjected to chemical analysis, plasma desorption mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. Chemical analysis of the purified hexamethyl LPS quantitated certain key chemical compositions. Plasma desorption mass spectrometry showed a molecular ion (M + CH2 + Na)+ at m/z 2360, which established the molecular formula and Mr to be C116H214N2O39P2 and 2323, respectively. Thus, it contained two each of glucosamine, 2-keto-3-deoxyoctonate, and phosphate; four beta-hydroxymyristates; one laurate; and one myristate. NMR spectroscopy confirmed the locations of the four ester-linked fatty acyl groups. Based on these results and the known structure of free lipid A, the complete structure of the deep-rough chemotype LPS from E. coli can now be presented with confidence. This is the first report of a successful purification to homogeneity and the characterization of the simplest of the LPS at the intact level. This study shows that the natural distribution of the lipid A moiety of LPS from E. coli D31m4 is hexaacyl/pentaacyl in a molar ratio of greater than 90:less than 10. Acid hydrolysis of LPS causes the formation of the lower homologues of the free lipid A.     

Gating charges in the activation and inactivation processes of the HERG channel

The hERG channel has a relatively slow activation process but an extremely fast and voltage-sensitive inactivation process. Direct measurement of hERG's gating current (Piper, D.R., A. Varghese, M.C. Sanguinetti, and M. Tristani-Firouzi. 2003. PNAS. 100:10534-10539) reveals two kinetic components of gating charge transfer that may originate from two channel domains. This study is designed to address three questions: (1) which of the six positive charges in hERG's major voltage sensor, S4, are responsible for gating charge transfer during activation, (2) whether a negative charge in the cytoplasmic half of S2 (D466) also contributes to gating charge transfer, and (3) whether S4 serves as the sole voltage sensor for hERG inactivation. We individually mutate S4's positive charges and D466 to cysteine, and examine (a) effects of mutations on the number of equivalent gating charges transferred during activation (z(a)) and inactivation (z(i)), and (b) sidedness and state dependence of accessibility of introduced cysteine side chains to a membrane-impermeable thiol-modifying reagent (MTSET). Neutralizing the outer three positive charges in S4 and D466 in S2 reduces z(a), and cysteine side chains introduced into these positions experience state-dependent changes in MTSET accessibility. On the other hand, neutralizing the inner three positive charges in S4 does not affect z(a). None of the charge mutations affect z(i). We propose that the scheme of gating charge transfer during hERG's activation process is similar to that described for the Shaker channel, although hERG has less gating charge in its S4 than in Shaker. Furthermore, channel domain other than S4 contributes to gating charge involved in hERG's inactivation process.