Computational studies of membrane channels

2005

Motivation. In this paper we analyze the electrostatic interactions in the open and closed states of the KcsA channel. We focus on the stability of the KWKWK…K sequence already proposed as a model for the permeation mechanism of the KcsA transmembrane channel. The fact that the channel can accommodate more than one ion in the pore is crucial to explain the high diffusion rate of K + ions observed. We also address the question of the accuracy of the force fields by comparing them to ab initio calculations. Method. On the basis of molecular dynamics calculations, we determine the electrostatic potential due to the protein embedded in its membrane using either AMBER6 force fields or quantum calculations conducted with GAUSSIAN 03 at the HF 6-31G(d) level. Conclusions. Both force field and quantum approaches show that water molecules located in the KWKWK…K sequence play a structuring role for the ions in the filter. When compared to the closed state situation, the open state conformation of the protein promotes very different behaviors of K + and water motions in the hydrophobic cavity and in the external mouth of the channel. Keywords. KcsA channel; ab initio; molecular dynamics; electrostatics. Abbreviations and notations K n , Potassium ion located in the n th site of the pore HF, Hartree-Fock W n , Water molecule located in the n th site of the pore DFT, Density functional theory MD, Molecular dynamics QM/MM, Quantum mechanics/molecular mechanics BD, Brownian dynamics 1 INTRODUCTION Research in ion channels has long had a strong experimental tradition with relatively little input from theory [1,2]. The reason for this imbalance between theory and experiment is usually attributed to the complexity of both the biological processes and the underlying molecular structures involved in their realization. Rapid advances at two fronts, namely determination of the tertiary structure of macromolecules from X-ray diffraction and NMR techniques and an exponential

Proceedings of the National Academy of Sciences, 2006

Potassium channels are necessary for a number of essential biological tasks such as the generation of action potentials and setting the resting membrane potential in cells, both of which require that these channels selectively permit the passage of potassium ions while suppressing the flow of other ions. Generally, this selectivity is attributed to a narrow stretch of the channel known as the selectivity filter. Over this stretch ions are dehydrated, and the backbone oxygen atoms of the protein mimic the ion's loss of coordination by water. However, channels are long pores with spatially distinct ion-binding sites that all must be traversed during ion permeation. We have shown that selectivity of mutant Kir3.2 (GIRK2) channels can be substantially amplified by introducing acidic residues into the cavity, a binding site below the selectivity filter. Here, we carry out electrostatic calculations on homology models to quantify the degree of stabilization that these mutations have on ions in the cavity. We then construct a multiion model of ion permeation to calculate the channel's permeability to potassium relative to sodium. This kinetic model uses rates derived from the electrostatic calculations and demonstrates that nonselective electrostatic stabilization of cations in the cavity can amplify channel selectivity independently of the selectivity filter. This nonintuitive result highlights the dependence of channel properties on the entire channel architecture and suggests that selectivity may not be fully understood by focusing solely on thermodynamic considerations of ion dehydration and the energetics of the selectivity filter.

Proteins-structure Function and Bioinformatics, 2003

The availability of structural information about biological ion channels provides an opportunity to gain a detailed understanding of the control of ion selectivity by biological systems. However, accomplishing this task by computer simulation approaches is very challenging. First, although the activation barriers for ion transport can be evaluated by microscopic simulations, it is hard to obtain accurate results by such approaches. Second, the selectivity is related to the actual ion current and not directly to the individual activation barriers. Thus, it is essential to simulate the ion currents and this cannot be accomplished at present by microscopic MD approaches. In order to address this challenge, we developed and refined an approach capable of evaluating ion current while still reflecting the realistic features of the given channel. Our method involves generation of semimacroscopic free energy surfaces for the channel/ions system and Brownian dynamics (BD) simulations of the corresponding ion current. In contrast to most alternative macroscopic models, our approach is able to reproduce the difference between the free energy surfaces of different ions and thus to address the selectivity problem. Our method is used in a study of the selectivity of the KcsA channel toward the K ؉ and Na ؉ ions. The BD simulations with the calculated free energy profiles produce an appreciable selectivity. To the best of our knowledge, this is the first time that the trend in the selectivity in the ion current is produced by a computer simulation approach. Nevertheless, the calculated selectivity is still smaller than its experimental estimate. Recognizing that the calculated profiles are not perfect, we examine how changes in these profiles can account for the observed selectivity. It is found that the origin of the selectivity is more complex than generally assumed. The observed selectivity can be reproduced by increasing the barrier at the exit and the entrance of the selectivity filter, but the necessary changes in the barrier approach the limit of the error in the PDLD/S-LRA calculations. Other options that can increase the selectivity are also considered, including the difference between the Na ؉ . . . Na ؉ and K ؉ . . . K ؉ interaction. However, this interesting effect does not appear to lead to a major difference in selectivity since the Na ؉ ions at the limit of strong interaction tend to move in a less concerted way than the K ؉ ions. Changes in the relative binding energies at the different binding sites are also not so effective in changing the selectivity. Finally, it is pointed out that using the calculated profiles as a starting point and forcing the model to satisfy different experimentally based constraints, should eventually provide more detailed understanding of the different complex factors involved in ion selectivity of biological channels. Proteins 2003;52:412-426.

Journal of Biological Chemistry, 2017

Journal of Biological Chemistry, 2006

The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. Curr. Opin. Struct. Biol. 14, 440-446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA and its chymotryptic derivative 1-125 KcsA reconstituted into native-like membranes differ from those exhibited by these proteins in detergent solution, the latter taken as an approximation of the mixed detergent-protein crystal conditions.

IEEE Transactions on Nanobioscience, 2005

Biophysical Journal, 2003

Interactions of Na 1 , K 1 , Rb 1 , and Cs 1 ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K 1 and Rb 1 , are similar in their interactions with the selectivity filter. In contrast, Na 1 ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs 1 ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs 1 ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.

Ion channels are proteins that are embedded within the lipid bilayer membranes of cells and some viruses, where their physiological function is to regulate the passage of small charged molecules in and out of the cell or its various organelles . Because of lipid dielectric properties, an ion needs 20 times more energy than an uncharged water molecule to pass through a biological membrane. This is why ion channels and pumps are necessary to transport charged molecules through cell membranes. Ion channels are mostly pore opening proteins which are fractions of a nanometre in radius and a few nanometres long. The simplest way to picture them is as hollow proteins that allow the transit of charged and neutral solutes, driven either by a concentration gradient or by the electric potential difference between the inner and the outer side of the membrane; or by both simultaneously. There are several families and sub-families of ion channels and new varieties are constantly reported. They can be classified by their permeability properties, their activation mechanism or simply by their size. However, these criteria are often interrelated. For instance, channels that perform the very specific function of translocating say, Ca 2+ ions, display a narrow aqueous pore, while channels that allow permeation by cations and anions are usually wider. A "narrow" protein channel pore is comparable to the unhydrated size of small inorganic ions like K + and Na + . This review focuses on the selective properties of wide channels, collectively known as mesoscopic channels because of their size. Transport through these channels is passive and multiionic, and it is mainly regulated by electrostatic interactions between protein ionizable residues and the permeating ions. Examples of mesoscopic channels are bacterial porins like OmpF from E. coli , the voltage-dependent anion channel (VDAC) of the mitochondria outer membrane [13], pore-opening toxins like the alphahemolysin channel secreted by S. Aureus and antibiotic peptides like Alamethicin . Most of them share structural motifs: for example, transmembrane pores consist largely Resum Els canals iònics de gran amplada i de conductància alta exerceixen una funció molt important en el cicle de vida de les cèllules. Permeten l'intercanvi de soluts neutres i carregats a través de la membrana cel·lular i regulen l'aportació de nutrients i la sortida de deixalles. Per a realitzar aquesta funció, els canals han de discriminar entre diferents espècies iòniques. Els canals mesoscòpics permeten el transport multiiònic passiu i generalment presenten una selectivitat moderada respecte a ions positius i negatius. Aquí revisem les metodologies més usades per a estimar quantitativament la selectivitat iònica. Entre elles, la mesura del potencial elèctric necessari per a anul·lar el corrent elèctric a través del canal en presència d'un gradient de concentració, cosa que es coneix com a potencial de corrent zero. Assenyalem els factors clau que cal tenir en compte per a una interpretació física correcta d'aquests experiments d'electrofisiologia.

Scientific Reports, 2019

Potassium channels selectivity filter (SF) conformation is modulated by several factors, including ionprotein and protein-protein interactions. Here, we investigate the SF dynamics of a single Trp mutant of the potassium channel KcsA (W67) using polarized time-resolved fluorescence measurements. For the first time, an analytical framework is reported to analyze the homo-Förster resonance energy transfer (homo-FRET) within a symmetric tetrameric protein with a square geometry. We found that in the closed state (pH 7), the W67-W67 intersubunit distances become shorter as the average ion occupancy of the SF increases according to cation type and concentration. The hypothesis that the inactivated SF at pH 4 is structurally similar to its collapsed state, detected at low K + , pH 7, was ruled out, emphasizing the critical role played by the S2 binding site in the inactivation process of KcsA. This homo-FRET approach provides complementary information to X-ray crystallography in which the protein conformational dynamics is usually compromised. Potassium channels are integral membrane proteins present in prokaryotic and eukaryotic organisms where they contribute to the control of potassium flow, cell volume, release of hormones and neurotransmitters, resting potential, excitability, and behavior. Under physiological conditions these molecules are highly selective, allowing the permeation of K + at near diffusion-limited rates, whereas Na + is effectively excluded from passing through 1. So far, the molecular basis of selectivity and permeation is still a matter of debate. KcsA is a proton-activated, voltage-modulated channel cloned from S. lividans and used as a prototypical protein on the biophysical studies of the K + channels due to its simple structure: four identical subunits around a central pore, each one comprising an N-terminal domain, two transmembrane segments and the cytosolic C-terminal section. The two transmembrane segments (TM1 and TM2) are connected by a pore region that contains a tilted short-helix (pore helix), two loops and an ion selectivity filter (SF) (Fig. 1A). The SF contains four putative K + binding sites delineated by the signature sequence TVGYG (Fig. 1B,C), clearly homologous to the more complex eukaryotic potassium channels and its conformational dynamics is mainly responsible for the permeation and the selectivity features of these membrane proteins 2,3. Based on the conformation of the intracellular bundle (intracellular gate or gate I) and the SF (gate II), the gating cycle of KcsA is defined by at least four kinetic states: closed/conductive (pH7, high K +), open/conductive (transient state found in high K + after the pH4 gating), open/inactivated (pH4, high K + , at steady state) and closed/inactivated (pH7, high K + , immediately after increasing the pH from 4 to 7) 4. The opening movement at gate I is allosterically coupled to changes in SF conformation, inducing the slow inactivation process, and consequently leading to a steady-state, very low open probability of the wild-type (WT) channel 5,6. The inactivation phenomenon after several milliseconds of activity occurs in a very similar way to eukaryotic channels 7,8. Additionally, X-ray crystallography data from the closed state revealed two different conformations of the SF: a conductive conformation (Fig. 1B,C) in the presence of sufficient amounts of permeant cations (e.g. K + , Rb + or Cs +), and a non-conductive or collapsed structure when in low concentration of permeant species or high quantities of blocking species such as Na + , with only the most external binding sites (S1 and S4) accessible to ion-protein interaction (Fig.

Journal of Molecular Biology, 2010

Potassium (K + ) channels are specialized membrane proteins able to facilitate and regulate the conduction of K + ions through cell membranes. Comprising five specific cation binding sites (S 0 to S 4 ) formed by the backbone carbonyl groups of conserved residues common to all K + channels, the narrow selectivity filter allows fast conduction of K + while being highly selective for K + ions over sodium (Na + ) ions. To extend our knowledge of the microscopic mechanism underlying selectivity in K + channels, we characterize the free energy landscapes governing the entry and translocation of a Na + or a K + ion from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K + ions in the pore and the 3-ion potential of mean force (PMF) is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the PMFs yields a number of important results. First, the free energy minima corresponding to configurations with the extracellular K + or Na + ion in the binding site S 0 or S 1 are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between the stable multi-ion configurations are generally higher for the Na + ion than for the K + ion, implying that the kinetics of ion conduction is slower when a Na + enters the pore. Third, the region corresponding to the binding site S 2 near the center of the narrow pore emerges as being the most selective for K + over Na + . In particular, while there is a stable minimum for K + in the site S 2 , the Na + ion faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near the binding site S 2 in the selectivity filter of K + channels.