Antimicrobial Peptides and Skin: A Paradigm of Translational Medicine

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic peptides with broad-spectrum microbicidal activity against both bacteria and fungi. In mammals, AMPs form the first line of host defense against infections and generally play an important role as effector agents of the innate immune system. The AMP era was born more than 6 decades ago when the first cationic cyclic peptide antibiotics, namely polymyxins and tyrothricin, found their way into clinical use. Due to the good clinical experience in the treatment of, for example, infections of mucus membranes as well as the subsequent understanding of mode of action, AMPs are now considered for treatment of inflammatory skin diseases and for improving healing of infected wounds. Based on the preclinical findings, including pathobiochemistry and molecular medicine, targeted therapy strategies are developed and first results indicate that AMPs influence processes of diseased skin. Importantly, in contrast to other antibiotics, AMPs do not seem to propagate the development of antibiotic-resistant micro-organisms. Therefore, AMPs should be tested in clinical trials for their efficacy and tolerability in inflammatory skin diseases and chronic wounds. Apart from possible fields of application, these peptides appear suited as an example of the paradigm of translational medicine for skin diseases which is today seen as a `two-way road' - from bench to bedside and backwards from bedside to bench. Copyright (c) 2012 S. Karger AG, Base

NaChBac: The Long Lost Sodium Channel Ancestor

In excitable cells, the main mediators of sodium conductance across membranes are voltage-gated sodium channels (Na(V)s). Eukaryotic Na(V)s are essential elements in neuronal signaling and muscular contraction and in humans have been causally related to a variety of neurological and cardiovascular channelopathies. They are complex heavily glycosylated intrinsic membrane proteins present in only trace quantities that have proven to be challenging objects of study. However, in recent years, a number of simpler prokaryotic sodium channels have been identified, with NaChBac from Bacillus halodurans being the most well-characterized to date. The availability of a bacterial Na(V) that is amenable to heterologous expression and functional characterization in both bacterial and mammalian systems has provided new opportunities for structure--function studies. This review describes features of NaChBac as an exemplar of this class of bacterial channels, compares prokaryotic and eukaryotic Na(V)s with respect to their structural organization, pharmacological profiling, and functional kinetics, and discusses how voltage-gated ion channels may have evolved to deal with the complex functional demands of higher organisms

Fever as a Cause of Hypophosphatemia in Patients with Malaria

Hypophosphatemia occurs in 40 to 60% of patients with acute malaria, and in many other conditions associated with elevations of body temperature. To determine the prevalence and causes of hypophosphatemia in patients with malaria, we retrospectively studied all adults diagnosed with acute malaria during a 12-year period. To validate our findings, we analyzed a second sample of malaria patients during a subsequent 10-year period. Serum phosphorus correlated inversely with temperature (n = 59, r = −0.62; P<0.0001), such that each 1°C increase in body temperature was associated with a reduction of 0.18 mmol/L (0.56 mg/dL) in the serum phosphorus level (95% confidence interval: −0.12 to −0.24 mmol/L [−0.37 to −0.74 mg/dL] per 1°C). A similar effect was observed among 19 patients who had repeat measurements of serum phosphorus and temperature. In a multiple linear regression analysis, the relation between temperature and serum phosphorus level was independent of blood pH, PCO2, and serum levels of potassium, bicarbonate, calcium, albumin, and glucose. Our study demonstrates a strong inverse linear relation between body temperature and serum phosphorus level that was not explained by other factors known to cause hypophosphatemia. If causal, this association can account for the high prevalence of hypophosphatemia, observed in our patients and in previous studies of patients with malaria. Because hypophosphatemia has been observed in other clinical conditions characterized by fever or hyperthermia, this relation may not be unique to malaria. Elevation of body temperature should be added to the list of causes of hypophosphatemia

Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

An unusual mechanism of ion channel regulation generates voltage-dependent gating in the absence of a canonical voltage-sensing domain

Determining Peptide Partitioning Properties via Computer Simulation

The transfer of polypeptide segments into lipid bilayers to form transmembrane helices represents the crucial first step in cellular membrane protein folding and assembly. This process is driven by complex and poorly understood atomic interactions of peptides with the lipid bilayer environment. The lack of suitable experimental techniques that can resolve these processes both at atomic resolution and nanosecond timescales has spurred the development of computational techniques. In this review, we summarize the significant progress achieved in the last few years in elucidating the partitioning of peptides into lipid bilayer membranes using atomic detail molecular dynamics simulations. Indeed, partitioning simulations can now provide a wealth of structural and dynamic information. Furthermore, we show that peptide-induced bilayer distortions, insertion pathways, transfer free energies, and kinetic insertion barriers are now accurate enough to complement experiments. Further advances in simulation methods and force field parameter accuracy promise to turn molecular dynamics simulations into a powerful tool for investigating a wide range of membrane active peptide phenomena

A Genome-Wide Linkage and Association Scan Reveals Novel Loci for Hypertension and Blood Pressure Traits

Hypertension is caused by the interaction of environmental and genetic factors. The condition which is very common, with about 18% of the adult Hong Kong Chinese population and over 50% of older individuals affected, is responsible for considerable morbidity and mortality. To identify genes influencing hypertension and blood pressure, we conducted a combined linkage and association study using over 500,000 single nucleotide polymorphisms (SNPs) genotyped in 328 individuals comprising 111 hypertensive probands and their siblings. Using a family-based association test, we found an association with SNPs on chromosome 5q31.1 (rs6596140; P<9×10−8) for hypertension. One candidate gene, PDC, was replicated, with rs3817586 on 1q31.1 attaining P = 2.5×10−4 and 2.9×10−5 in the within-family tests for DBP and MAP, respectively. We also identified regions of significant linkage for systolic and diastolic blood pressure on chromosomes 2q22 and 5p13, respectively. Further family-based association analysis of the linkage peak on chromosome 5 yielded a significant association (rs1605685, P<7×10−5) for DBP. This is the first combined linkage and association study of hypertension and its related quantitative traits with Chinese ancestry. The associations reported here account for the action of common variants whereas the discovery of linkage regions may point to novel targets for rare variant screening

Conformational Changes and Slow Dynamics through Microsecond Polarized Atomistic Molecular Simulation of an Integral Kv1.2 Ion Channel

Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins—not to mention numerous applications in drug design. Here, we present a full 1 µs atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120° rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation (∼35°) of the extracellular end of all S4 segments is present also in a reference 0.5 µs simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 310 helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4–lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5–1 µs). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations

Gramicidin tryptophans mediate formamidinium-induced channel stabilization.

Compared with alkali metal cations, formamidinium ions stabilize the gramicidin A channel molecule in monoolein bilayers (Seoh and Busath, 1993a). A similar effect is observed with N-acetyl gramicidin channel molecules in spite of the modified forces at the dimeric junction (Seoh and Busath, 1993b). Here we use electrophysiological measurements with tryptophan-to-phenylalanine-substituted gramicidin analogs to show that the formamidinium-induced channel molecule stabilization is eliminated when the four gramicidin tryptophans are replaced with phenylalanines in gramicidin M-. This suggests that the stabilization is mediated by the tryptophan side chains. Tryptophan residues 9, 13, and 15 must cooperate to produce the effect because replacement of any one of the three with phenylalanine significantly reduces stabilization; replacement of Trp-11 with phenylalanine causes negligible decrease in stabilization. In addition, formamidinium-related current-voltage supralinearity and open-channel noise are absent with gramicidin M-. When the lipid bilayer was formed with monoolein ether rather than monoolein ester, the channel lifetimes were reduced markedly and, at low voltage and relative to those in KCl solution, were decreased by a factor of 2, whereas the open-channel noise was unaffected and the current-voltage relation was only modestly affected. These results suggest that formamidinium modifies the state of the tryptophan side chains, which, in turn, affects channel lifetime, current-voltage supralinearity, and open-channel noise through interactions with water or lipid headgroup atoms including the lipid ester carbonyl

The permeation properties of small organic cations in gramicidin A channels.

The conductance properties of organic cations in single gramicidin A channels were studied using planar lipid bilayers. From measurements at 10 mM and at 27 mV the overall selectivity sequence was found to be NH4+ > K+ > hydrazinium > formamidinium > Na+ > methylammonium, which corresponds to Eisenman polyatomic cation sequence X'. Methylammonium and formamidinium exhibit self block, suggesting multiple occupancy and single filing. Formamidinium has an apparent dissociation constant (which is similar to those of alkali metal cations) for the first ion being 22 mM from the Eadie-Hofstee plot (G0 vs. G0/C), 12 mM from the rate constants of a three-step kinetic model. The rate-limiting step for formamidinium is translocation judging from supralinear I-V relations at low concentrations. 1 M formamidinium solutions yields exceptionally long single channel lifetimes, 20-fold longer than methylammonium, which yields lifetimes similar to those found with alkali metal cations. The average lifetime in formamidinium solution significantly decreases with increasing voltage up to 100 mV but is relatively voltage independent between 100 and 200 mV. At lower voltages (< or = 100 mV), the temperature and concentration dependences of the average lifetime of formamidinium were steep. At very low salt concentrations (0.01 M, 100 mV), there was no significant difference in average lifetime from that formed with 0.01 M methylammonium or hydrazinium. We conclude that formamidinium very effectively stabilizes the dimeric channel while inside the channel and speculate that it does so by affecting tryptophan-reorientation or tryptophan-lipid interactions at binding sites