ACID-SENSING ION CHANNELS: functional and molecular characterization in putative nociceptors, and modulation by nerve injury and serine protease

Résumé Les canaux ioniques ASICs (acid-sensing ion channels) appartiennent à la famille des canaux ENaC/Degenerin. Pour l'instant, quatre gènes (1 à 4) ont été clonés dont certains présentent des variants d'épissage. Leur activation par une acidification rapide du milieu extracellulaire génère un courant entrant transitoire essentiellement sodique accompagné pour certains types d'ASICs d'une phase soutenue. Les ASICs sont exprimés dans le système nerveux, central (SNC) et périphérique (SNP). On leur attribue un rôle dans l'apprentissage, la mémoire et l'ischémie cérébrale au niveau central ainsi que dans la nociception (douleur aiguë et inflammatoire) et la méchanotransduction au niveau périphérique. Toutefois, les données sont parfois contradictoires. Certaines études suggèrent qu'ils sont des senseurs primordiaux impliqués dans la détection de l'acidification et la douleur. D'autres études suggèrent plutôt qu'ils ont un rôle modulateur inhibiteur dans la douleur. De plus, le fait que leur activation génère majoritairement un courant transitoire alors que les fibres nerveuses impliquées dans la douleur répondent à un stimulus nocif avec une adaptation lente suggère que leurs propriétés doivent être modulés par des molécules endogènes. Dans une première partie de ma thèse, nous avons abordé la question de l'expression fonctionnelle des ASICs dans les neurones sensoriels primaires afférents du rat adulte pour clarifier le rôle des ASICs dans les neurones sensoriels. Nous avons caractérisé leurs propriétés biophysiques et pharmacologiques par la technique du patch-clamp en configuration « whole-cell ». Nous avons pu démontrer que près de 60% des neurones sensoriels de petit diamètre expriment des courants ASICs. Nous avons mis en évidence trois types de courant ASIC dans ces neurones. Les types 1 et 3 ont des propriétés compatibles avec un rôle de senseur du pH alors que le type 2 est majoritairement activé par des pH inférieurs à pH6. Le type 1 est médié par des homomers de la sous-unité ASIC1 a qui sont perméables aux Ca2+. Nous avons étudié leur co-expression avec des marqueurs des nocicepteurs ainsi que la possibilité d'induire une activité neuronale suite à une acidification qui soit dépendante des ASICs. Le but était d'associer un type de courant ASIC avec une fonction potentielle dans les neurones sensoriels. Une majorité des neurones exprimant les courants ASIC co-expriment des marqueurs des nocicepteurs. Toutefois, une plus grande proportion des neurones exprimant le type 1 n'est pas associée à la nociception par rapport aux types 2 et 3. Nous avons montré qu'il est possible d'induire des potentiels d'actions suite à une acidification. La probabilité d'induction est proportionnelle à la densité des courants ASIC et à l'acidité de la stimulation. Puis, nous avons utilisé cette classification comme un outil pour appréhender les potentielles modulations fonctionnelles des ASICs dans un model de neuropathie (spared nerve injury). Cette approche fut complétée par des expériences de «quantitative RT-PCR ». En situation de neuropathie, les courants ASIC sont dramatiquement changés au niveau de leur expression fonctionnelle et transcriptionnelle dans les neurones lésés ainsi que non-lésés. Dans une deuxième partie de ma thèse, suite au test de différentes substances sécrétées lors de l'inflammation et l'ischémie sur les propriétés des ASICs, nous avons caractérisé en détail la modulation des propriétés des courants ASICs notamment ASIC1 par les sérines protéases dans des systèmes d'expression recombinants ainsi que dans des neurones d'hippocampe. Nous avons montré que l'exposition aux sérine-protéases décale la dépendance au pH de l'activation ainsi que la « steady-state inactivation »des ASICs -1a et -1b vers des valeurs plus acidiques. Ainsi, l'exposition aux serine protéases conduit à une diminution du courant quand l'acidification a lieu à partir d'un pH7.4 et conduit à une augmentation du courant quand l'acidification alleu à partir d'un pH7. Nous avons aussi montré que cette régulation a lieu des les neurones d'hippocampe. Nos résultats dans les neurones sensoriels suggèrent que certains courants ASICs sont impliqués dans la transduction de l'acidification et de la douleur ainsi que dans une des phases du processus conduisant à la neuropathie. Une partie des courants de type 1 perméables au Ca 2+ peuvent être impliqués dans la neurosécrétion. La modulation par les sérines protéases pourrait expliquer qu'en situation d'acidose les canaux ASICs soient toujours activables. Résumé grand publique Les neurones sont les principales cellules du système nerveux. Le système nerveux est formé par le système nerveux central - principalement le cerveau, le cervelet et la moelle épinière - et le système nerveux périphérique -principalement les nerfs et les neurones sensoriels. Grâce à leur nombreux "bras" (les neurites), les neurones sont connectés entre eux, formant un véritable réseau de communication qui s'étend dans tout le corps. L'information se propage sous forme d'un phénomène électrique, l'influx nerveux (ou potentiels d'actions). A la base des phénomènes électriques dans les neurones il y a ce que l'on appelle les canaux ioniques. Un canal ionique est une sorte de tunnel qui traverse l'enveloppe qui entoure les cellules (la membrane) et par lequel passent les ions. La plupart de ces canaux sont normalement fermés et nécessitent d'être activés pour s'ouvrire et générer un influx nerveux. Les canaux ASICs sont activés par l'acidification et sont exprimés dans tout le système nerveux. Cette acidification a lieu notamment lors d'une attaque cérébrale (ischémie cérébrale) ou lors de l'inflammation. Les expériences sur les animaux ont montré que les canaux ASICs avaient entre autre un rôle dans la mort des neurones lors d'une attaque cérébrale et dans la douleur inflammatoire. Lors de ma thèse je me suis intéressé au rôle des ASICs dans la douleur et à l'influence des substances produites pendant l'inflammation sur leur activation par l'acidification. J'ai ainsi pu montrer chez le rat que la majorité des neurones sensoriels impliqués dans la douleur ont des canaux ASICs et que l'activation de ces canaux induit des potentiels d'action. Nous avons opéré des rats pour qu'ils présentent les symptômes d'une maladie chronique appelée neuropathie. La neuropathie se caractérise par une plus grande sensibilité à la douleur. Les rats neuropathiques présentent des changements de leurs canaux ASICs suggérant que ces canaux ont une peut-être un rôle dans la genèse ou les symptômes de cette maladie. J'ai aussi montré in vitro qu'un type d'enryme produit lors de l'inflammation et l'ischémie change les propriétés des ASICs. Ces résultats confirment un rôle des ASICs dans la douleur suggérant notamment un rôle jusque là encore non étudié dans la douleur neuropathique. De plus, ces résultats mettent en évidence une régulation des ASICs qui pourrait être importante si elle se confirmait in vivo de part les différents rôles des ASICs. Abstract Acid-sensing ion channels (ASICs) are members of the ENaC/Degenerin superfamily of ion channels. Their activation by a rapid extracellular acidification generates a transient and for some ASIC types also a sustained current mainly mediated by Na+. ASICs are expressed in the central (CNS) and in the peripheral (PNS) nervous system. In the CNS, ASICs have a putative role in learning, memory and in neuronal death after cerebral ischemia. In the PNS, ASICs have a putative role in nociception (acute and inflammatory pain) and in mechanotransduction. However, studies on ASIC function are somewhat controversial. Some studies suggest a crucial role of ASICs in transduction of acidification and in pain whereas other studies suggest rather a modulatory inhibitory role of ASICs in pain. Moreover, the basic property of ASICs, that they are activated only transiently is irreconcilable with the well-known property of nociception that the firing of nociceptive fibers demonstrated very little adaptation. Endogenous molecules may exist that can modulate ASIC properties. In a first part of my thesis, we addressed the question of the functional expression of ASICs in adult rat dorsal root ganglion (DRG) neurons. Our goal was to elucidate ASIC roles in DRG neurons. We characterized biophysical and pharmacological properties of ASIC currents using the patch-clamp technique in the whole-cell configuration. We observed that around 60% of small-diameter sensory neurons express ASICs currents. We described in these neurons three ASIC current types. Types 1 and 3 have properties compatible with a role of pH-sensor whereas type 2 is mainly activated by pH lower than pH6. Type 1 is mediated by ASIC1a homomultimers which are permeable to Ca 2+. We studied ASIC co-expression with nociceptor markers. The goal was to associate an ASIC current type with a potential function in sensory neurons. Most neurons expressing ASIC currents co-expressed nociceptor markers. However, a higher proportion of the neurons expressing type 1 was not associated with nociception compared to type 2 and -3. We completed this approach with current-clamp measurements of acidification-induced action potentials (APs). We showed that activation of ASICs in small-diameter neurons can induce APs. The probability of AP induction is positively correlated with the ASIC current density and the acidity of stimulation. Then, we used this classification as a tool to characterize the potential functional modulation of ASICs in the spared nerve injury model of neuropathy. This approach was completed by quantitative RT-PCR experiments. ASICs current expression was dramatically changed at the functional and transcriptional level in injured and non-injured small-diameter DRG neurons. In a second part of my thesis, following an initial screening of the effect of various substances secreted during inflammation and ischemia on ASIC current properties, we characterized in detail the modulation of ASICs, in particular of ASIC1 by serine proteases in a recombinant expression system as well as in hippocampal neurons. We showed that protease exposure shifts the pH dependence of ASIC1 activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in the current response if ASIC1 is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provided evidence that this bi-directional regulation of ASIC1a function also occurs in hippocampal neurons. Our results in DRG neurons suggest that some ASIC currents are involved in the transduction of peripheral acidification and pain. Furthermore, ASICs may participate to the processes leading to neuropathy. Some Ca 2+-permeable type 1 currents may be involved in neurosecretion. ASIC modulation by serine proteases may be physiologically relevant, allowing ASIC activation under sustained slightly acidic conditions.

A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina

Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K+ currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Navs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Nav1.7-specific inhibitor, ProTxII, allowed the isolation of Nav1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Nav1.7 and Nav1.8 currents. The redistribution of Nav1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L-/-). SNS-Nedd4L-/- mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Nav1.7 and Nav1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Navs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Capacity of MIMO Channels: asymptotic evaluation under correlated fading

This paper investigates the asymptotic uniform power allocation capacity of frequency nonselective multiple-inputmultiple-output channels with fading correlation at either thetransmitter or the receiver. We consider the asymptotic situation,where the number of inputs and outputs increase without boundat the same rate. A simple uniparametric model for the fadingcorrelation function is proposed and the asymptotic capacity perantenna is derived in closed form. Although the proposed correlationmodel is introduced only for mathematical convenience, itis shown that its shape is very close to an exponentially decayingcorrelation function. The asymptotic expression obtained providesa simple and yet useful way of relating the actual fadingcorrelation to the asymptotic capacity per antenna from a purelyanalytical point of view. For example, the asymptotic expressionsindicate that fading correlation is more harmful when arising atthe side with less antennas. Moreover, fading correlation does notinfluence the rate of growth of the asymptotic capacity per receiveantenna with high Eb /N0.

Enhanced MDIR receiver for space-time dispersive channels

A particular property of the matched desiredimpulse response receiver is introduced in this paper, namely,the fact that full exploitation of the diversity is obtained withmultiple beamformers when the channel is spatially and timelydispersive. This particularity makes the receiver specially suitablefor mobile and underwater communications. The new structureprovides better performance than conventional and weightedVRAKE receivers, and a diversity gain with no needs of additionalradio frequency equipment. The baseband hardware neededfor this new receiver may be obtained through reconfigurabilityof the RAKE architectures available at the base station. Theproposed receiver is tested through simulations assuming UTRAfrequency-division-duplexing mode.

Joint array combining and MLSE for single-user receivers in multipath Gaussian multiuser channels

The well-known structure of an array combiner along with a maximum likelihood sequence estimator (MLSE) receiveris the basis for the derivation of a space-time processor presentinggood properties in terms of co-channel and intersymbol interferencerejection. The use of spatial diversity at the receiver front-endtogether with a scalar MLSE implies a joint design of the spatialcombiner and the impulse response for the sequence detector. Thisis faced using the MMSE criterion under the constraint that thedesired user signal power is not cancelled, yielding an impulse responsefor the sequence detector that is matched to the channel andcombiner response. The procedure maximizes the signal-to-noiseratio at the input of the detector and exhibits excellent performancein realistic multipath channels.

Mapping out customer segments and marketing channels in the Finnish Homelift market

Työn tavoitteena oli saada selville ketkä ovat uuden tuotteen, eli kotihissin asiakkaat. Mitkä ovat tuotteen mahdollisuudet Suomen markkinoilla ja onko markkinoilla odotettavissa kasvua, sekä miten tähän mahdolliseen kasvuun päästäisiin käsiksi. Kotihissin ominaisuudet vaikuttavat siihen, että se on tarkoitettu asennettavaksi yksityisiin pientaloihin. Uuden tuotteen ansiosta KONE voi liittää nykyiseen vahvaan toimialaansa, eli kerrostaloihin, uuden aluevaltauksen; Pientalot. Tämä 'laajentuminen' tuo mukanaan kuluttajamarkkinoiden haasteet. Markkinatilanteen selvittämiseen käyttin jo olemassa olevaa tietoa hyväkseni; sanomalehdistä markkinatutkimuksiin. Asiakasryhmät kartoitin tutustumalla messutapahtumissa saatuihin kontakteihin. Päätutkimusmenetelmänä käytin haastatteluja. Niihin osallistui kaikkiaan 14 vastaajaa. He vasatsivat kysymyksiin ostomotiiveista ja tuotteesta. Eräs haastattelujen tärkeimmistä teemoista liittyi yhteydenpito- ja jakelukanaviin, joista 12 vastaajaa antoivat mielipiteitään. Työni tulokset viittaavat siihen, että markkinatilanne näyttää positiiviselta KONEen kannalta. Tutkimuksesta sain selville kotihissin asiakassegmentit ja myös miten nämä potentiaaliset asiakkaat saadaan tehokkaimmin tavoitettua.

Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.

Derivation of an observer model adapted to irregular signals based on convolution channels.

Anthropomorphic model observers are mathe- matical algorithms which are applied to images with the ultimate goal of predicting human signal detection and classification accuracy across varieties of backgrounds, image acquisitions and display conditions. A limitation of current channelized model observers is their inability to handle irregularly-shaped signals, which are common in clinical images, without a high number of directional channels. Here, we derive a new linear model observer based on convolution channels which we refer to as the "Filtered Channel observer" (FCO), as an extension of the channelized Hotelling observer (CHO) and the nonprewhitening with an eye filter (NPWE) observer. In analogy to the CHO, this linear model observer can take the form of a single template with an external noise term. To compare with human observers, we tested signals with irregular and asymmetrical shapes spanning the size of lesions down to those of microcalfications in 4-AFC breast tomosynthesis detection tasks, with three different contrasts for each case. Whereas humans uniformly outperformed conventional CHOs, the FCO observer outperformed humans for every signal with only one exception. Additive internal noise in the models allowed us to degrade model performance and match human performance. We could not match all the human performances with a model with a single internal noise component for all signal shape, size and contrast conditions. This suggests that either the internal noise might vary across signals or that the model cannot entirely capture the human detection strategy. However, the FCO model offers an efficient way to apprehend human observer performance for a non-symmetric signal.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Inverse convection problem of simultaneous estimation of two boundary heat fluxes in parallel plate channels

This paper deals with the use of the conjugate gradient method of function estimation for the simultaneous identification of two unknown boundary heat fluxes in parallel plate channels. The fluid flow is assumed to be laminar and hydrodynamically developed. Temperature measurements taken inside the channel are used in the inverse analysis. The accuracy of the present solution approach is examined by using simulated measurements containing random errors, for strict cases involving functional forms with discontinuities and sharp-corners for the unknown functions. Three different types of inverse problems are addressed in the paper, involving the estimation of: (i) Spatially dependent heat fluxes; (ii) Time-dependent heat fluxes; and (iii) Time and spatially dependent heat fluxes.

Integrating the export distribution channels after an acquisition

This thesis is based on studying integration synergies in a merger or acquisition situation with the interest in distribution channel integration. M&As seem to be forever popular and nowadays companies often use them as a certain kind of strategy to develop their business. M&As have attracted research for decades and also distribution in M&As has been found interesting. Moreover, research often concentrates to the horizontal M&A´s potential synergies that emerge in the integration process and so is the case also in this study as the core issue is to study the integration of the distribution channels and its potential synergies. This study concentrates on a single case, an acquisition which took place in 2011. The case consists of two Finnish companies operating in the same business field. Both of the companies are very export orientated, which gives this study its export view. As the companies operate in the same field this acquisition falls into the categorization of horizontal acquisition. The objective of the thesis is to study how the export channels could be integrated after an acquisition? This research question is divided again to three sub-questions asking how the distribution channels of the acquirer and acquired company are organized, what is pursued by the post-acquisition distribution channel integration and what are the integration challenges especially from the logistics point-of-view. The framework was built from the basis of the literature used in this thesis. The framework combines M&A process and distribution channels to a one united model which presents the progress of this thesis. The study was carried out as a qualitative research and as a holistic single case study. The data used in the research includes two interviews, other material from the case companies and also material of the companies collected by the author independently from different sources. There were many motives for the acquisition as usual. From the research results one can find that the integration process is still very ongoing and the synergies have not yet been fully discovered but also they are there to be found. The concentration of the research was in the export distribution which proved to be meaningful since the exports markets of the case companies were found to be quite complementary. The research results brought up also other issues concerning the post-acquisition integration process rather than the distribution channels and export. M&As are always a risky business. The final result can never be predicted. No matter how good the merging companies look on paper the practice is not the same. The acquisition process of this case has not ended yet and there lies potential synergy benefits to be discovered if enough effort is used to go through the process right.

Connexin domains relevant to the chemical gating of gap junction channels

Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons), each formed by the radial arrangement of six connexin (Cx) proteins. Connexins span the bilayer four times (M1-M4) and have both amino- and carboxy-termini (NT, CT) at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2) and one inner (IL) loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO2 treatment) or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO2, whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL2) of Cx38 was as sensitive to CO2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO2 sensitivity, but replacement of 5 arginines (R) with sparagines (N) at the beginning of CT (C1) greatly enhanced the CO2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C1 maintain the CO2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning of IL1 and positive residues of either C1 or IL2. Open and closed channels would result from IL1-C1 and IL1-IL2 interactions, respectively

Integrating Use of Customer Information Throughout Multiple Sales Channels to Create Customer Competence

Customer knowledge management (CKM) practices enable organizations to create customer competence with systematic use of customer information that is integrated throughout the organization. Nonetheless, organizations are not able to fully exploit the vast amount of data available. Previous research on use of customer information is limited especially in a multichannel environment. The aim of this study was to identify the main obstacles for utilizing customer information efficiently across multiple sales channels. The study was conducted as a single case study in order to gain deeper understanding of the research problem. The empirical findings indicate that lack of CKM practices and a common goal are major challenges obstructing effective utilization of customer information. Furthermore, decentralized organizational structure and insufficient analytical skills create obstacles for information sharing and capabilities to process information and create new knowledge. The implications of the study suggest that in order to create customer competence organizations should shift their focus from technology to the organizational factors affecting use of information and implement CKM practices throughout the organization.

Connexin hemichannels and cell-cell channels: comparison of properties

Connexin46 (Cx46) forms functional hemichannels in the absence of contact by an apposed hemichannel and we have used these hemichannels to study gating and permeation at the single channel level with high time resolution. Using both cell-attached and -excised patch configurations, we find that single Cx46 hemichannels exhibit some properties expected of half of a gap junction channel, as well as novel properties. Cx46 hemichannels have a large unitary conductance (~300 pS) and a relatively large pore as inferred from permeability to TEA. Both monovalent cations and anions can permeate, but cations are substantially more permeable. The open channel conductance shows marked inward rectification in symmetric salts. We find that the conductance and permeability properties of Cx46 cell-cell channels can be explained by the series addition of two hemichannels. These data suggest that the pore structures of unapposed hemichannels and cell-cell channels are conserved. Also like cell-cell channels, unapposed Cx46 hemichannels are closed by elevated levels of H+ or Ca2+ ions on the cytoplasmic face. Closure occurs in excised patches indicating that the actions of these agents do not require a soluble cytoplasmic factor. Fast (<0.5 ms) application of H+ to either side of the open hemichannel causes an immediate small reduction in unitary conductance followed by complete closure with latencies that are dependent on H+ concentration and side of application; sensitivity is much greater to H+ on the cytoplasmic side. Closure by cytoplasmic H+ does not require that the hemichannel be open. Thus, H+ ions readily permeate Cx46 hemichannels, but at high enough concentration close them by acting at a cytoplasmic site(s) that causes a conformational change resulting in complete closure. Extracellular H+ may permeate to act on the cytoplasmic site or act on a lower affinity extracellular site. Thus, the unapposed hemichannel is a valuable tool in addressing fundamental questions concerning the operation of gap junction channels that are difficult to answer by existing methods. The ability of Cx46, and perhaps other connexins, to form functional unapposed hemichannels that are opened by moderate depolarization may represent an unexplored role of connexins as mediators of transport across the plasma membrane.