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MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.

The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl − channels and Cl − /H + antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Glu ex , could explain the transport cycle of CLC-type Cl − /H + antiporters. However, the presence of multiple conformations ( Up , Middle , and Down ) of the Glu ex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae . Thus, we aimed to investigate further the heterogeneity of Glu ex -conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Glu ex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the “ Midlow ” conformation. We also found that an extra anion can be located above the external Cl − -binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl − -binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Glu ex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.

CLC-ec1 is a Cl−/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4–5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

CLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

Background Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare form of infantile-onset leukodystrophy. The disorder is caused primarily by mutations of MLC1 that leads to a series of phenotypic outcomes including vacuolation of myelin and astrocytes, subcortical cysts, brain edema, and macrocephaly. Recent studies have indicated that functional interactions among MLC1, GlialCAM, and ClC-2 channels play key roles in the regulation of neuronal, glial and vascular homeostasis. However, the physiological role of MLC1 in cellular homeostatic communication remains poorly understood. In the present study, we investigated the cellular function of MLC1 and its effects on cell–cell interactions. Methods MLC1-dependent cellular morphology and motility were analyzed by using confocal and live cell imaging technique. Biochemical approaches such as immunoblotting, co-immunoprecipitation, and surface biotinylation were conducted to support data. Results We found that the altered MLC1 expression and localization led to a great alteration in cellular morphology and motility through actin remodeling. MLC1 overexpression induced filopodia formation and suppressed motility. And, MLC1 proteins expressed in patient-derived MLC1 mutants resulted in trapping in the ER although no changes in morphology or motility were observed. Interestingly knockdown of Mlc1 induced Arp3-Cortactin interaction, lamellipodia formation, and increased the membrane ruffling of the astrocytes. These data indicate that subcellular localization of expressed MLC1 at the plasma membrane is critical for changes in actin dynamics through ARP2/3 complex. Thus, our results suggest that misallocation of pathogenic mutant MLC1 may disturbs the stable cell-cell communication and the homeostatic regulation of astrocytes in patients with MLC.

This article introduces the history and the long-term goals of the Korea Brain Initiative, which is centered on deciphering the brain functions and mechanisms that mediate the integration and control of brain functions that underlie decision-making. The goal of this initiative is the mapping of a functional connectome with searchable, multi-dimensional, and information-integrated features. The project also includes the development of novel technologies and neuro-tools for integrated brain mapping. Beyond the scientific goals this grand endeavor will ultimately have socioeconomic ramifications that not only facilitate global collaboration in the neuroscience community, but also develop various brain science-related industrial and medical innovations.

Additional file 4: Figure S4. Examine the infection efficiency and the effect of MLC1 on the matured primary astrocytes. (A) Knockdown of MLC1 in mouse primary astrocytes was performed by viral infection and was confirmed via Western Blotting with anti-MLC1 antibody. GFP expression levels were analyzed to evaluate the efficiency of AAV infection. Similar GFP expression level in the astrocytes treating with either shMlc1 or the scrambled shMlc1 (shScr) virus indicates that the infection efficiency was similar. Actin was used as loading control. (B and C) To verify the maturation of primary astrocytes, cells were stained with anti-S100β antibody (magenta). Empty and filled arrowheads in the magnified image indicate fibrous actin bundles and branched actin networks, respectively.

The CLC family proteins are involved in a variety of cellular processes, where chloride homeostasis needs to be controlled. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Recent studies have revealed that the conformational heterogeneity of the critical glutamate residue, Gluex could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations of the Gluex has been suggested from combined structural snapshots of two different CLC antiporters. Thus, we aimed to investigate the presence of these three intermediate conformations in CLC-ec1, the most deeply studied CLC at both functional and structural levels. By comparing crystal structures of E148D, E148A mutant and wildtype CLC-ec1 with varying anion concentrations, we suggest that the Gluex indeed take at least three distinct conformational states in a single CLC antiporter, CLC-ec1.

A subclass of bacterial CLC anion-transporting proteins, phylogenetically distant from long-studied CLCs, was recently shown to be specifically up-regulated by F - . We establish here that a set of randomly selected representatives from this “CLC F ” clade protect Escherichia coli from F - toxicity, and that the purified proteins catalyze transport of F - in liposomes. Sequence alignments and membrane transport experiments using 19 F NMR, osmotic response assays, and planar lipid bilayer recordings reveal four mechanistic traits that set CLC F proteins apart from all other known CLCs. First, CLC F s lack conserved residues that form the anion binding site in canonical CLCs. Second, CLC F s exhibit high anion selectivity for F - over Cl - . Third, at a residue thought to distinguish CLC channels and transporters, CLC F s bear a channel-like valine rather than a transporter-like glutamate, and yet are F - /H + antiporters. Finally, F - /H + exchange occurs with 1∶1 stoichiometry, in contrast to the usual value of 2∶1.

Animal locomotion is mediated by a sensory system referred to as proprioception. Defects in the proprioceptive coordination of locomotion result in uncontrolled and inefficient movements. However, the molecular mechanisms underlying proprioception are not fully understood. Here, we identify two transient receptor potential cation (TRPC) channels, trp-1 and trp-2, as necessary and sufficient for proprioceptive responses in C. elegans head steering locomotion. Both channels are expressed in the SMDD neurons, which are required and sufficient for head bending, and mediate coordinated head steering by sensing mechanical stretches due to the contraction of head muscle and orchestrating dorsal head muscle contractions. Moreover, the SMDD neurons play dual roles to sense muscle stretch as well as to control muscle contractions. These results demonstrate that distinct locomotion patterns require dynamic and homeostatic modulation of feedback signals between neurons and muscles., Author summary Proprioception provides the nervous system with feedback about body posture in animals and is essential for the generation of coherent locomotive behaviors, such as walking, running, or crawling. However, little is known about the identity of proprioceptive receptors that sense body movement to regulate locomotion and the extent to which proprioception modulates sensorimotor coordination. Here, we analyze the molecular mechanisms that control head steering locomotion of Caenorhabditis elegans. We show that this movement is regulated by the transient receptor potential cation (TRPC) channels TRP-1 and TRP-2 and the SMDD proprioceptive neurons. We observe that mutant animals for both channels are defective in head steering locomotion and that ectopic expression of TRP-1 or TPR-2 in a C. elegans chemosensory neuron confers head bending–dependent responses, suggesting roles for these channels in proprioception. We also find that SMDD neurons are both necessary and sufficient to generate head steering locomotion via the two channels. Moreover, we demonstrate that the proprioceptive system mediates locomotion coordination by desynchronizing activities in motor systems. We conclude that two TRPC channels in collaboration with the proprioceptive receptor SMDD neurons control head steering in worms during forward locomotion.

CLC-ec1, a bacterial homologue of the CLC family's transporter subclass, catalyzes transmembrane exchange of Cl(-) and H(+). Mutational analysis based on the known structure reveals several key residues required for coupling H(+) to the stoichiometric countermovement of Cl(-). E148 (Glu(ex)) transfers protons between extracellular water and the protein interior, and E203 (Glu(in)) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H(+) transport while preserving Cl(-) transport. We tested the role of Glu(in) by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H(+)/Cl(-) exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H(+) coupling and severely inhibit Cl(-) flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H(+) transport with substantial Cl(-) transport. Influence on H(+) transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Glu(in) is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl(-) movements to bring about Cl(-)/H(+) exchange.

Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has been widely used in northern and eastern Asia to treat diverse diseases. Here, we investigated the anti-senescent effects of ethanolic extracts of Dalbergia odorifera (EEDO) in ultraviolet (UV) B-irradiated skin cells. EEDO significantly inhibited UVB-induced senescence of human keratinocytes in a concentration-dependent manner, concomitant with inhibition of reactive oxygen species (ROS) generation. UVB-induced increases in the levels of p53 and p21, biomarkers of cellular senescence, were almost completely abolished in the presence of EEDO. Sativanone, a major constituent of EEDO, also attenuated UVB-induced senescence and ROS generation in keratinocytes, indicating that sativanone is an indexing (marker) molecule for the anti-senescence properties of EEDO. Finally, treatment of EEDO to mice exposed to UVB significantly reduced ROS levels and the number of senescent cells in the skin. Thus, EEDO confers resistance to UVB-induced cellular senescence by inhibiting ROS generation in skin cells.

Background & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Methods: The patch-clamp technique for whole-cell recording was used in cultured or single interstitial cells of Cajal. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Results: Electrophysiological and pharmacological properties of the nonselective cation channel in interstitial cells of Cajal were the same as those of TRPM7. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of TRPM7 messenger RNA and protein in mouse small intestine. Treatment of primary cultured interstitial cells of Cajal with TRPM7-specific small interfering RNA resulted in inhibition of pacemaking activity. Conclusions: TRPM7 is required for intestinal pacemaking. The protein is a likely potential target for pharmacological treatment of motor disorders of the gut.

Ankyrin-repeat family A protein (ANKRA) was originally cloned in mouse as an interacting protein to megalin, a member of low-density lipoprotein receptor superfamily. Here, we report that the isolation of rat ANKRA as a new binding partner for the α-subunit of rat large-conductance Ca2+-activated K+channel (rSlo). We mapped the binding region of each protein by using yeast two-hybrid and in vitro binding assays. ANKRA expressed together with rSlo channels were colocalized near the plasma membrane and coimmunoprecipitated in transfected cells. We also showed that BKCachannel in rat cerebral cortex coprecipitated with rANKRA and colocalized in cultured rat hippocampal neuron. Although the coexpression of ANKRA did not affect the surface expression of rSlo, the gating kinetics of rSlo channel was significantly altered and the effects were highly dependent on the intracellular calcium. These results indicate that ANKRA could modulate the excitability of neurons by binding directly to endogenous BKCachannel and altering its gating kinetics in a calcium-dependent manner.

The incidence of acute mastoiditis has declined dramatically during the postantibiotic era. Even so, antibiotic-resistant or unusual pathogens can still cause this disease entity. At our hospital, we documented an increase in antibiotic-resistant and atypical pathogens such as Actinomyces spp. and Mycobacterium tuberculosis. In this paper, we discuss the optimal diagnosis and treatment strategy for acute mastoiditis, and we describe our retrospective review of 13 patients with mastoiditis who were treated at our hospital from 1988 through 1998. Eight of these patients recovered following treatment with intravenous antibiotics, with or without myringotomy, and five who had complications of disease were managed surgically. Among these five, one developed chronic otitis media and one developed cholesteatoma 3 years later. For patients with acute mastoiditis, we emphasize the need to be aware of any unusual pathogens that do not respond to empiric antibiotic therapy.

Cl(-)/H(+) antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl(-), Br(-), I(-), NO3(-) and SCN(-), but they seem to be very selective against F(-). The recent discovery of a new CLC clade of F(-)/H(+) antiporters, which are highly selective for F(-) over Cl(-), led us to investigate the mechanism of Cl(-)-over-F(-) selectivity by a CLC Cl(-)/H(+) antiporter, CLC-ec1. By subjecting purified CLC-ec1 to anion transport measurements, electrophysiological recording, equilibrium ligand-binding studies and X-ray crystallography, we show that F(-) binds in the Cl(-) transport pathway with affinity similar to Cl(-) but stalls the transport cycle. Examination of various mutant antiporters implies a 'lock-down' mechanism of F(-) inhibition, in which F(-), by virtue of its unique hydrogen-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.

Mutagenesis, functional analysis, and crystal structures identify a watery tunnel through which protons enter the interior of a Cl−/H+ antiport protein involved in acid resistance of enteric bacteria., Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H+-gated Cl− channels and Cl−/H+ antiporters. Transmembrane H+ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl− ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein–water boundary of the interfacial region, leads to severe slowing of the Cl−/H+ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Gluin physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin., Author Summary Chloride-proton antiport proteins of the “CLC” superfamily are transmembrane proteins that form homodimers and are used for myriad physiological purposes, all requiring the coordinated movements of Cl− anions and H+ cations in opposite directions across biological membranes. While the pathway for Cl− ions through CLC antiporters is known, we currently have only indirect glimpses of how protons navigate their way through these membrane-embedded proteins. By combining mechanistic and structural approaches, we identify a proton-access pathway in a bacterial Cl−/H+ antiporter that allows intracellular protons to enter the protein interior and engage in the coupled antiport mechanism. We conclude that E202, a highly conserved glutamate residue, serves to organize water molecules and guide protons to the adjacent glutamate E203 (known as “Gluin”), a critical residue for the antiport mechanism.

CLC-ec1 is a bacterial CLC Cl-/H+ exchanger that catalyzes 2:1 exchange of Cl- and H+. E148 is the pH-dependent external gate of both Cl- and H+ transport and E203 mediates proton transport from intracellular solution to the protein interior. Substitution of non-protonatable residues on either glutamate uncouples the exchanger by completely abolishing H+ transport. However, it is not clear how protons in the intracellular solution gain access to E203, which is buried under a cytoplasmic “lid” formed by the protein's N-terminal helix. Truncation of the N-terminal 29 residues removes this lid and preserves 2:1 coupling of Cl- and H+ and absolute transport rate. This result implies that proton transport from intracellular solution to E203 is not rate-limiting in the transport cycle, and that protons are somehow facilitated in their movement to the buried E203. In order to discover proton facilitators, we examined polar residues near E203. Nonpolar mutants of Q207, S446 and R403 only slightly inhibit H+-transport rate. However, mutations on the proximate glutamate, E113, and disruption of a nearby salt-bridge (E117I and R209I) decrease H+-transport rate ∼5-fold. These residues appear to form a water-filled conduit for proton access to E203. Interestingly, nonpolar mutants of E202, located near the dimer interface and near to intracellular solution, show 30-100-fold reduced H+-transport rates compared to wildtype. All mutations here preserve H+-coupled Cl- transport, although those with severely reduced rates display somewhat higher Cl-/H+ stoichiometry, indicative of Cl- slippage. One or two crystallographic water molecules were found between E203 and E202. We propose that these water molecules form an access pathway for H+ from bulk water, which is blocked by bulky, hydrophobic amino acids at E202.

The CLC-ec1 is a bacterial homologue of the CLC Cl-/H+ exchanger that catalyzes 2:1 exchange of Cl- and H+. Functionally intact monomers form a dimer in bilayer membranes. Gating-glutamate (or Gluex, E148) is the pH-dependent external gate of both Cl- and H+ transport and proton-glutamate (or Gluin, E203) mediates proton-transport from intracellular solution to the protein interior. Mutations on either glutamate with non-protonable amino acids abolish H+ transport across the membrane. However, it is still puzzling how protons in the intracellular solution could be transferred to the proton-gluatamate forming a salt bridge with arginine (R28) in swapped N-terminal domain from neighboring subunit, which structurally isolates the proton-glutamate from the intracellular solution. In order to find functional facilitators for proton transport, we have been examining several residues near E203. Salt-bridge breaking mutants, R28L, Q and E do not alter the functional activity at all. Moreover, N-terminal truncation mutants (Delta16 and Delta29), which remove an apparent “lid” covering the proton-glutamate, preserve intact Cl-/H+ exchanger activity. Mutations on two proximate glutamates (E113L and E202L) decrease H+ transport rate without changing Cl- turnover rate. Currently, we are investigating functional and structural changes of multiple mutations.

The CLC-ec1 is a bacterial homologue of the transporter-type CLC subclass that catalyzes transmembrane exchange of Cl− and H+. Gluex (E148 in CLC-ec1) is the pH-dependent external gate of both Cl− and H+ transport. The central tyrosine, Y445 (Tyrcen) coordinates the central Cl− ion and acts as the internal gate of the Cl− pathway. Gluin (E203) is remotely located to the internal Cl− gate and acts as the internal proton-transfer residue that delivers protons from the internal aqueous solution to the protein interior. Wild type CLC-ec1 has the pH-dependent Cl- flux: as the pH increased, the transport rate of Cl- is decreased. It has been shown that mutations on the Gluex lead to the loss of pH-dependency of Cl− flux while on the Gluin retain relatively intact pH-dependency. We therefore examined mutations of Gluex and their functional consequences on the pH-dependency. Mutations with neutral amino acids, A or Q (E148A or E148Q) completely lose H+ movement as well as pH-dependency of Cl− transport. E148H mutant also fail to transport H+, but strikingly reverses pH-dependent transport of Cl−, as does the doubly mutated transporter, E148H/E203H. These data support the idea that the pH-dependency is mainly came from the Gluex not Gluin. Currently, we are investigating the effect of other mutants having various ranges of pKa value and their structural and functional consequences.

It has been suggested that the large conductance Ca(2)+-activated K(+) channel contains one or more domains known as regulators of K(+) conductance (RCK) in its cytosolic C terminus. Here, we show that the second RCK domain (RCK2) is functionally important and that it forms a heterodimer with RCK1 via a hydrophobic interface. Mutant channels lacking RCK2 are nonfunctional despite their tetramerization and surface expression. The hydrophobic residues that are expected to form an interface between RCK1 and RCK2, based on the crystal structure of the bacterial MthK channel, are well conserved, and the interactions of these residues were confirmed by mutant cycle analysis. The hydrophobic interaction appears to be critical for the Ca(2+)-dependent gating of the large conductance Ca(2+)-activated K(+) channel.

The proteins that 'pump' neurotransmitters into neurons, clearing the synapse after a nerve impulse, are central players in coherent brain function and are targets of many psychotropic drugs. Two groups now endeavor to resolve a fundamental controversy about how these proteins work. The results shed new light on the controversy but do not end it.

Objectives The cell cycle must be involved in cell proliferation of the epithelium of middle ear cholesteatoma. Cyclins and cyclin-dependent kinase (CDK) complexes have important regulatory roles during cell cycle progression. Cyclin-CDK complexes are in turn regulated by the cyclin-dependent kinase inhibitors (CDKIs), which generally inhibit cell cycle progression. One of the important CDKI members is p27Kip1. The goal of this study is to evaluate the expression of p27Kip1 and Ki-67, a proliferation marker, in cholesteatoma and in the skin of the external ear canal. Methods The expressions of p27Kip1 and Ki-67 in cholesteatoma epithelium (n = 20) and ear canal epithelium (n = 7) were investigated by an immunohistochemical technique. Results In cholesteatoma epithelium specimens, the expression of p27Kip1 was observed from the parabasal layer to the granular layer, but not in the basal layer. Ki-67 was expressed dominantly in the basal and parabasal cell layers. Their expressions tend to be increased compared with their expressions in the normal ear canal skin. The expression pattern of the proliferation marker Ki-67 in the epithelial layers of two groups was inversely related to the expression of p27Kip1. Conclusions In cholesteatoma, the expressions of CDKI and Ki-67 were both increased in this study. The ability to inhibit proliferative activity was also increased in the cholesteatoma epithelium. The expression pattern of the proliferation marker Ki-67 in the epithelial layers was inversely related to the expression of p27Kip1. Not only is the proliferation activity increased, but also the ability to inhibit hyperproliferation is increased in the cholesteatoma epidermis. Despite increased proliferative activity in the cholesteatoma epidermis, epithelial cells still retain the capability to prevent cell cycle arrest by means of p27Kip1.

Two putative homologues of large conductance Ca2+-activated K+ channel alpha-subunit gene (slowpoke or slo) were revealed by C. elegans genome sequencing. One of the two genes, F08B12.3 (Ce-slo-2), shows a relatively low amino acid sequence similarity to other Slo sequences and lacks key functional motifs, which are important for calcium and voltage sensing. However, its overall structure and regions of homology, which are conserved in all Slo proteins, suggest that Ce-SLO-2 should belong to the Slo channel family. We have cloned a full-length cDNA of the Ce-slo-2, which encodes a protein containing six putative transmembrane segments with a K+-selective pore and a large C-terminal cytosolic domain. Green fluorescent protein (GFP) and whole-mount immunostaining analyses revealed that Ce-slo-2 is specifically expressed in neuronal cells at the nerve ring, at the ventral nerve cord of the mid-body, and at the tail region. We have also identified a putative human counterpart of Ce-slo-2 from a human brain EST database, which shows a stretch of highly conserved amino acid residues. Northern blot and mRNA dot blot analyses revealed a strong and specific expression in brain and skeletal muscle. Taken together, our data suggest that Ce-slo-2 may constitute an evolutionarily conserved gene encoding a potassium channel that has specific functions in neuronal cells.

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H+-gated Cl− channels and Cl−/H+ antiporters. Transmembrane H+ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl− ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl−/H+ antiport rate. The x-ray crystal structure of E202Y, the most severely affected mutant, suggests that the aromatic ring of Y202 makes cross-subunit hydrophobic interaction with an I201 sidechain from neighboring subunit, which physically blocks the interfacial pathway. This mechanism is functionally supported by the minimal effect of E202Y mutation in a monomeric variant of the transporter, which does not have such an interaction. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin.

Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.