Encapsulation of DNA into hydroxyapatite (HAp) has been investigated using a rational approach that involves computer simulation and experimental techniques. The temporal evolution of the radial distribution functions derived from atomistic molecular dynamics simulations of Ca

A conserved tryptophan uniquely present in the voltage-sensing domain of voltage-gated proton channels is surprisingly crucial to four channel-defining properties.

No abstract

The voltage-gated proton channel 1 (H V 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. H V 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an H V 1 channel. In the present study, we report the existence of an H V 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative H V 1 orthologues in various polyneopteran insects. To confirm that these putative H V 1 orthologues were functional channels, we studied the H V 1 channel of Nicoletia phytophila (NpH V 1), an insect of the Zygentoma order, in more detail. NpH V 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pHand voltage-dependent gating. Interestingly, NpH V 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels.

The H V 1 voltage-gated proton (H V 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of H V 1 is Zn 2+. Externally applied ZnCl 2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn 2+ and Cd 2+ on H V 1 from Nicoletia phytophila, NpH V 1. We introduced mutations at potential Zn 2+ coordination sites and measured Zn 2+ inhibition in different extracellular pH, with Zn 2+ concentrations up to 1000 lM. Zn 2+ inhibition in NpH V 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn 2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn 2+ in the S3-S4 linker. A simple Hodgkin Huxley model of NpH V 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpH V 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development. Abbreviations Asp145, aspartate at position 145; g H,max , maximal proton conductance; g H,max /10, 10% of the maximal proton conductance; g H-V, proton conductance-voltage relationship; hH V 1, human voltage-gated proton channel; H V 1, voltage-gated proton channel 1; NpH V 1, Nicoletia phytophila voltage-gated proton channel 1; pH i , pH inside the cell; pH o , pH outside the cell; TM, transmembrane domain; TMA, tetramethylammonium; TSA, Transcriptome Shotgun Assembly; V rev , reversal potential; V threshold , threshold potential.