Warren J. Gallin

Calcium regulation of gene expression in neurons: the mode of entry matters.

Ca2+ entry into neurons is one of the major effectors of stimulus-induced physiological change. Ca2+ can enter neurons through a number of different voltage-gated and ligand-gated channels. Depending on the route of entry, Ca2+ stimulates distinct intracellular signaling pathways, which activate different sets of genes, resulting in alternative physiological outcomes for the cell. These recent results suggest that the specific effect of a single biochemical second messenger can vary as a consequence of its route of entry into the cell.

Calcium Channel Structural Determinants of Synaptic Transmission between Identified Invertebrate Neurons

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaearequires the expression of a C-terminal splice variant of theLymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.

In the First Extracellular Domain of E-cadherin, Heterophilic Interactions, but Not the Conserved His-Ala-Val Motif, Are Required for Adhesion

The classical cadherins, definitive proteins of the cadherin superfamily, are characterized functionally by their ability to mediate calcium-dependent cell aggregationin vitro. To test hypothetical mechanisms of adhesion, we have constructed two mutants of the chicken E-cadherin protein, one with the highly conserved His-Ala-Val (HAV) sequence motif reversed to Val-Ala-His (VAH), the other lacking the first extracellular domain (EC1). The inversion of HAV to VAH has no effect on the capacity of E-cadherin to mediate adhesion. Deletion of EC1 completely eliminates the ability of E-cadherin to mediate homophilic adhesion, but the deletion mutant is capable of adhering heterophilically to both unmutated E-cadherin and to the HAV/VAH mutant. These results demonstrate that the conserved HAV sequence motif is not involved in cadherin-mediated adhesion as has been suggested previously and supports the idea that in the context of the cell surface, cadherin-mediated cell-cell adhesion involves an interaction of EC1 with other domains of the cadherin extracellular moiety and not the “linear zipper” model, which posits trans interactions only between EC1 on apposing cell surfaces.

VKCDB: Voltage-gated potassium channel database

BackgroundThe family of voltage-gated potassium channels comprises a functionally diverse group of membrane proteins. They help maintain and regulate the potassium ion-based component of the membrane potential and are thus central to many critical physiological processes. VKCDB (V oltage-gated potassium [K] C hannel D ataB ase) is a database of structural and functional data on these channels. It is designed as a resource for research on the molecular basis of voltage-gated potassium channel function.DescriptionVoltage-gated potassium channel sequences were identified by using BLASTP to search GENBANK and SWISSPROT. Annotations for all voltage-gated potassium channels were selectively parsed and integrated into VKCDB. Electrophysiological and pharmacological data for the channels were collected from published journal articles. Transmembrane domain predictions by TMHMM and PHD are included for each VKCDB entry. Multiple sequence alignments of conserved domains of channels of the four Kv families and the KCNQ family are also included. Currently VKCDB contains 346 channel entries. It can be browsed and searched using a set of functionally relevant categories. Protein sequences can also be searched using a local BLAST engine.ConclusionsVKCDB is a resource for comparative studies of voltage-gated potassium channels. The methods used to construct VKCDB are general; they can be used to create specialized databases for other protein families. VKCDB is accessible at http://vkcdb.biology.ualberta.ca.

VKCDB: voltage-gated K+ channel database updated and upgraded

The Voltage-gated K+ Channel DataBase (VKCDB) (http://vkcdb.biology.ualberta.ca) makes a comprehensive set of sequence data readily available for phylogenetic and comparative analysis. The current update contains 2063 entries for full-length or nearly full-length unique channel sequences from Bacteria (477), Archaea (18) and Eukaryotes (1568), an increase from 346 solely eukaryotic entries in the original release. In addition to protein sequences for channels, corresponding nucleotide sequences of the open reading frames corresponding to the amino acid sequences are now available and can be extracted in parallel with sets of protein sequences. Channels are categorized into subfamilies by phylogenetic analysis and by using hidden Markov model analyses. Although the raw database contains a number of fragmentary, duplicated, obsolete and non-channel sequences that were collected in early steps of data collection, the web interface will only return entries that have been validated as likely K+ channels. The retrieval function of the web interface allows retrieval of entries that contain a substantial fraction of the core structural elements of VKCs, fragmentary entries, or both. The full database can be downloaded as either a MySQL dump or as an XML dump from the web site. We have now implemented automated updates at quarterly intervals.

A naturally occurring omega current in a Kv3 family potassium channel from a platyhelminth

BackgroundVoltage-gated ion channels are membrane proteins containing a selective pore that allows permeable ions to transit the membrane in response to a change in the transmembrane voltage. The typical selectivity filter in potassium channels is formed by a tetrameric arrangement of the carbonyl groups of the conserved amino-acid sequence Gly-Tyr-Gly. This canonical pore is opened or closed by conformational changes that originate in the voltage sensor (S4), a transmembrane helix with a series of positively charged amino acids. This sensor moves through a gating pore formed by elements of the S1, S2 and S3 helices, across the plane of the membrane, without allowing ions to pass through the membrane at that site. Recently, synthetic mutagenesis studies in the Drosophila melanogaster Shaker channel and analysis of human disease-causing mutations in sodium channels have identified amino acid residues that are integral parts of the gating-pore; when these residues are mutated the proteins allow a non-specific cation current, known as the omega current, to pass through the gating-pore with relatively low selectivity.ResultsThe N.at-Kv3.2 potassium channel has an unusual weak inward rectifier phenotype. Several mutations of two amino acids in the voltage sensing (S4) transmembrane helix change the phenotype to a typical delayed rectifier. The inward rectifier channels (wild-type and mutant) are sensitive to 4-aminopyridine (4-AP) but not tetra-ethyl ammonium (TEA), whereas the delayed rectifier mutants are sensitive to TEA but not 4-AP. The inward rectifier channels also manifest low cation selectivity. The relative selectivity for different cations is sensitive to specific mutations in the S4 helix,ConclusionN.at-Kv3.2, a naturally occurring potassium channel of the Kv3 sequence family, mediates ion permeation through a modified gating pore, not the canonical, highly selective pore typical of potassium channels. This channel has evolved to yield qualitatively different ion permeability when compared to all other members of this gene family.

Burn healing in organ cultures of embryonic chicken skin: a model system

Burn wounds cause complex damage to the skin. Unlike simple cuts, burns cause a graded damage at the margin of the wound and leave biochemically complex debris in the wound, two factors that complicate the process of wound healing. To develop an in vitro system for studying cellular responses to burns in whole tissue, we have applied a standardized burn wound to organ cultures of embryonic chicken skin and evaluated cellular changes in response to the burn damage. This simplified system is not subject to uncontrolled infection and does not involve angiogenesis and granulation. Thus, these cultures provide a simplified model of how cells of the dermis and epidermis in and around a burn wound respond to heat damage early in the process of healing.

Computational identification of residues that modulate voltage sensitivity of voltage-gated potassium channels

BackgroundStudies of the structure-function relationship in proteins for which no 3D structure is available are often based on inspection of multiple sequence alignments. Many functionally important residues of proteins can be identified because they are conserved during evolution. However, residues that vary can also be critically important if their variation is responsible for diversity of protein function and improved phenotypes. If too few sequences are studied, the support for hypotheses on the role of a given residue will be weak, but analysis of large multiple alignments is too complex for simple inspection. When a large body of sequence and functional data are available for a protein family, mature data mining tools, such as machine learning, can be applied to extract information more easily, sensitively and reliably. We have undertaken such an analysis of voltage-gated potassium channels, a transmembrane protein family whose members play indispensable roles in electrically excitable cells.ResultsWe applied different learning algorithms, combined in various implementations, to obtain a model that predicts the half activation voltage of a voltage-gated potassium channel based on its amino acid sequence. The best result was obtained with a k-nearest neighbor classifier combined with a wrapper algorithm for feature selection, producing a mean absolute error of prediction of 7.0 mV. The predictor was validated by permutation test and evaluation of independent experimental data. Feature selection identified a number of residues that are predicted to be involved in the voltage sensitive conformation changes; these residues are good target candidates for mutagenesis analysis.ConclusionMachine learning analysis can identify new testable hypotheses about the structure/function relationship in the voltage-gated potassium channel family. This approach should be applicable to any protein family if the number of training examples and the sequence diversity of the training set that are necessary for robust prediction are empirically validated. The predictor and datasets can be found at the VKCDB web site [1].

DEVELOPMENT AND MAINTENANCE OF BILE CANALICULI IN VITRO AND IN VIVO

The apical surfaces of hepatocytes are specialized to form the boundaries of the bile canaliculi. The canaliculi function to secrete and concentrate components of the bile and to transport the bile out of the interior of the hepatic parenchymal tissue to the epithelium‐lined bile ducts. Failure of the canaliculi to form and function properly can lead to biliary stasis or release of bile components into the bloodstream, both potentially life‐threatening situations. Experimental analysis of canaliculus development and function has been undertaken in a number of experimental systems, ranging in complexity from intact animals to isolated hepatocyte cell cultures. These approaches each have inherent advantages and disadvantages for studying the various aspects of canaliculus development and function. This article summarizes what is known about how the functional components of the canaliculus develop and the directions that current experimental approaches are leading in analyzing this process. Studies of model epithelial systems have begun to define how interactions between components of the cytoskeleton and plasma membrane regulate the structure of polarized plasma membranes. These results are also discussed in terms of the bile canaliculus. Microsc. Res. Tech. 39:406–412, 1997.

Development of bile canaliculi between chicken embryo liver cells in vivo and in vitro

The development of an organized network of bile canaliculi is essential for the normal functioning of the liver. We have characterized bile canaliculus development in situ from Days 3-19 and in vitro in cultured hepatocyte monolayers using electron microscopical and immunofluorescent staining with antibodies that specifically recognize antigens of the bile canaliculus. Although the liver first forms as a discrete epithelial bud of endodermal tissue at stage 12-14 (45-53 h after laying), canaliculi were first detected by our antibodies at low levels in 4-day embryos and at high levels in stage 27 (5 days after laying) and later embryos. During Days 4, 5, and 6 the canaliculi near the periphery of the rudiment do not stain while canaliculi in central areas, closer to the gut, are strongly stained. During this transition period the ultrastructure of the canaliculi in the peripheral regions is also less developed than the central canaliculi where the antigens appear. By 7 days post laying, canaliculi throughout the entire liver rudiment express the marker antigens equally and have the ultrastructural characteristics of mature, functional canaliculi. Cells prepared from liver of embryos of 11 days incubation and grown in monolayer culture reformed discernible canalicular specializations, as determined by immunofluorescent staining and electron microscopy, but only transiently (for 1 to 3 days after plating). Not all of the antigens were expressed or polarized in these cultures. The capacity of the embryonic parenchymal cells to develop and maintain polarity appears to depend on factors possibly including age-dependent changes in the cells themselves, interactions with other cell types or extracellular matrix, or the shape of the cells.