Acid-sensing ion channels : modulation by serine-proteases and involvement in acid-induced action potential generation in hippocampal neurons

SUMMARY Acid-sensing ion channels (ASICs) are non-voltage gated sodium channels. They are activated by rapid extracellular acidification and generate an inactivating inward current. Four ASIC genes have been cloned: ASIC1, 2, 3 and 4, with variants a and b for ASIC1and AS1C2. ASICs are expressed in neurons of the central (CNS) and peripheral nervous system (PNS). In the CNS, ASICs have a role in learning, memory, as well as in neuronal death in ischemia. In the PNS, ASICs are involved in the perception of acid-induced pain, as well as in mechanoperception. In one part of my thesis project, we addressed the question of the mechanism of regulation of ASIC1 a by the serine protease trypsin at the molecular level. Trypsin modifies the function of ASIC1 a but not of ASIC1b. In order to identify the channel region responsible for this effect, we created chimeras between ASIC1 a and 1b. Subsequently, to identify the exact trypsin target(s), we mutated predicted trypsin sites in the region identified by the chimera. In the second part of a project, we investigated the role of ASICs at the cellular level, in neuronal signaling. Using the whole-cell patch clamp in hippocampal neuronal culture, we studied the potential involvement of ASICs in action potential (AP) generation. In the first part of the thesis work, we showed that trypsin modifies ASIC1a function: it shifts the pH activation and the steady-state inactivation curve towards more acidic values and accelerates the time course of the channel recovery from inactivation. We also showed that trypsin cleaves ASIC1a and that the functional effect and a channel cleavage correlate. In the inactivated state, channels cannot be modified by trypsin. Cleavage occurs in a channel region that is also important for inactivation of all ASICs; a part of this region is critical for the inhibition of ASIC1 a by the spider toxin Psalmotoxin1. In the second part of the thesis work, we showed that ASIC activity can modulate AP generation. ASIC activity by itself can induce trains of APs. In situations in which this activity by itself is not sufficient to induce APs, it can contribute to AP generation. During high neuronal activity, ASIC activity can block already existing trains of APs. In conclusion, depending on the activity of neuron in a particular moment, ASICs can differently modulate AP generation; they can induce, facilitate or inhibit APs. We also showed that trypsin changes the capability of ASICs to modulate AP generation by shifting the pH dependence to more acidic values, which adapts channel gating to pH conditions which may occur in pathological conditions such as ischemia. Our finding that trypsin modifies ASIC1 a function identifies a novel pharmacological tool, and proposes a mechanism of ASIC1a regulation that may have a physiological importance. The identification of the exact site of trypsin action gives insight to the molecular mechanisms of ASIC regulation. This work proposes a role in modulation of AP generation for ASICs in the CNS. RESUME Les canaux ASIC sont les canaux ioniques activ��s par l'acidification rapide extracellulaire. Activ��s, ils g��n��rent un courant entrant qui inactive en pr��sence de stimulus acide. Quatre g��nes ASIC ont ��t�� clon��s, ASIC1, 2, 3 et 4, avec les variants a et b pour ASIC1 et 2. Les ASICs sont exprim��s dans les neurones du syst��me nerveux central (SNC) et p��riph��rique (SNP). Dans le SNC, les ASIC ont un r��le dans le m��moire, apprentissage et la mort neuronale dans t'isch��mie. Dans le SNP, ils ont un r��le dans la perception de la douleur et m��chanosensation. Dans une partie de mon projet de th��se, nous avons ��tudi�� les m��canismes de la r��gulation d'ASIC1a par la s��rine-prot��ase trypsine au niveau mol��culaire. La trypsine modifie la fonction d'ASIC1a et pas ASIC1b. Nous avons cr���� les chim��res entre ASIC1 a et 1 b, afin d'identifier la r��gion du canal responsable pour l'effet. Pour identifier le(s) site(s) exactes de l'action de la trypsine, nous avons mut�� les sites potentiels de la trypsine dans la r��gion identifi��e par les chim��res. Dans la deuxi��me partie du projet, nous avons ��tudi�� le r��le des ASICs au niveau cellulaire. En utilisant la technique du patch clamp dans les cultures des neurones de l'hippocampe, nous avons ��tudi�� l'implication des ASICs dans la g��n��ration des potentiels d'action (PA). Nous avons montr�� que la trypsine agit sur le canal ASIC1a ; elle d��cale l'activation et �� steady-state �� inactivation vers les valeurs plus acides, et elle raccourcit le temps du �� recovery �� du canal. La trypsine coupe ASIC1a sur le r��sidu K145 et l'effet fonctionnel et la coupure corr��lent. Nous avons identifi�� la r��gion du canal responsable pour l'inactivation de tous les ASICs ; une partie de cette r��gion est responsable pour ['inhibition d'ASIC1 a par la Psalmotoxinel . Nous avons montr�� que les ASICs peuvent moduler la g��n��ration des PAs. L'activit�� des ASICs peut induire les trains des PAs. Quand l'activit�� des ASICs n'est pas suffisante pour induire le PA, elle peut contribuer �� sa g��n��ration. Pendant l'activit�� neuronale forte, l'activit�� des ASICs peut bloquer les trains des PAs qui existent d��j��. En conclusion, d��pendant de l'activit�� neuronale, les ASICs peuvent moduler la g��n��ration des PAs diff��remment ; ils peuvent induire, faciliter ou inhiber les PAs. La trypsine change la capacit�� des ASICs de moduler les PAs. Apr��s l'action de la trypsine, les ASICs peuvent moduler la g��n��ration des PAs dans les conditions l��g��rement acides, suivies par les fluctuations du pH acide, qui peuvent exister dans l'isch��mie. Le fait que la trypsine agit sur ASIC1a d��finit l'outil pharmacologique et propose le m��canisme de la r��gulation d'ASICI a qui pourrait avoir l'importance physiologique. L'identification du site de l'action de la trypsine ��claircit les m��canismes mol��culaires de la r��gulation des ASICs. Cette ��tude propose un r��le des ASICs dans la modulation de la g��n��ration des PAs. R��sum�� pour le public large Les neurones sont les cellules de syst��me nerveux dont la fonction est la signalisation. Comme toutes les autres cellules, les neurones ont une membrane qui s��pare l'int��rieur du milieu ext��rieur. Cette membrane est imperm��able pour des particules charg��es (ions). Dans cette membrane existent les prot��ines sp��cifiques, �� canaux ��, qui permettent le transport des ions d'un c��t�� de la membrane �� l'autre, comme r��ponse aux stimuli diff��rents. Ce transport des ions �� travers la membrane g��n��re un courant, qu'on peut mesurer. Ce courant est la base de la communication entre les neurones, ou, ce qu'on appelle la signalisation neuronale. Quand ce courant est suffisamment grand, il permet la g��n��ration du potentiel d'action, qui est le message principal de communication neuronale. Les canaux ASIC (acid-sensing ion channel), que nous ��tudions dans le laboratoire, sont activ��s par les acides. Les acides sont rel��ch��s dans beaucoup de situations dans le syst��me nerveux. Les ASIC ont ��t�� d��couverts r��cemment (en 1996), et nous ne connaissons pas encore tr��s bien toutes les fonctions de ces canaux. Nous savons qu'ils ont un r��le dans le m��moire, apprentissage, la sensation de la douleur et l'infarctus c��r��bral. Dans la premi��re partie de ce projet de th��se, nous avons voulu mieux comprendre comment fonctionnent ces canaux. Pour faire ��a, nous avons ��tudi�� la r��gulation des ASICs par une prot��ine, trypsine, qui coupe le canal ASIC. Nous avons ��tudi�� ou exactement la trypsine coupe le canal et quels effets ��a produit sur la fonction du canal. Dans la deuxi��me partie du projet de th��se, nous avons voulu mieux conna��tre comment le canal fonctionne au niveau de la cellule, comment il interagit avec les autres canaux et si il a un r��le dans la g��n��ration des potentiels d'action. Nous avons pu montrer que la trypsine change la fonction du canal, ce qui lui permet de fonctionner diff��remment. Nous avons aussi d��termin�� ou exactement ta trypsine coupe le canal. Au niveau de la cellule, nous avons montr�� que les ASIC peuvent moduler la g��n��ration des potentiels d'action, ��tant, d��pendant de l'activit�� du neurone, soit activateurs, soit inhibiteurs. La trypsine est une mol��cule qui peut ��tre lib��r��e dans le syst��me nerveux pendant certaines conditions, comme l'infarctus c��r��bral. A cause de ��a, les connaissances que la trypsine agit sur le anal ASIC pourraient ��tre important physiologiquement. La connaissance de l'endroit exacte ou la trypsine coupe le canal nous aide �� mieux comprendre la relation structure-fonction du canal. La modulation de la g��n��ration des potentiels d'actions par les ASIC indique que ces canaux peuvent avoir un r��le important dans la signalisation neuronale.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.

Guard cell-specific calcium sensitivity of high density and activity SV/TPC1 channels.

The slow vacuolar (SV) channel, a Ca2+-regulated vacuolar cation conductance channel, in Arabidopsis thaliana is encoded by the single-copy gene AtTPC1. Although loss-of-function tpc1 mutants were reported to exhibit a stoma phenotype, knowledge about the underlying guard cell-specific features of SV/TPC1 channels is still lacking. Here we demonstrate that TPC1 transcripts and SV current density in guard cells were much more pronounced than in mesophyll cells. Furthermore, the SV channel in motor cells exhibited a higher cytosolic Ca2+ sensitivity than in mesophyll cells. These distinct features of the guard cell SV channel therefore probably account for the published stomatal phenotype of tpc1-2.

Sodium channels and mammalian sensory mechanotransduction.

BACKGROUND: Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear. RESULTS: Here we show that deleting β and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro. CONCLUSION: Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane.

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca(2+) channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca(2+) and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca(2+)channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.

A unified treatment of optimum pilot overhead in multipath fading channels

The optimization of the pilot overhead in single-user wireless fading channels is investigated, and the dependence of this overhead on various system parameters of interest (e.g., fading rate, signal-to-noise ratio) is quantified. The achievable pilot-based spectral efficiency is expanded with respect to the fading rate about the no-fading point, which leads to an accurate order expansion for the pilot overhead. This expansion identifies that the pilot overhead, as well as the spectral efficiency penalty with respect to a reference system with genie-aided CSI (channel state information) at the receiver, depend on the square root of the normalized Doppler frequency. It is also shown that the widely-used block fading model is a special case of more accurate continuous fading models in terms of the achievable pilot-based spectral efficiency. Furthermore, it is established that the overhead optimization for multiantenna systems is effectively the same as for single-antenna systems with the normalized Doppler frequency multiplied by the number of transmit antennas.

Non-peaky signals in wideband fading channels: achievable bit rates and optimal bandwidth

In the context of fading channels it is well established that, with a constrained transmit power, the bit rates achievable by signals that are not peaky vanish as the bandwidth grows without bound. Stepping back from the limit, we characterize the highest bit rate achievable by such non-peaky signals and the approximate bandwidth where that apex occurs. As it turns out, the gap between the highest rate achievable without peakedness and the infinite-bandwidth capacity (with unconstrained peakedness) is small for virtually all settings of interest to wireless communications. Thus, although strictly achieving capacity in wideband fading channels does require signal peakedness, bit rates not far from capacity can be achieved with conventional signaling formats that do not exhibit the serious practical drawbacks associated with peakedness. In addition, we show that the asymptotic decay of bit rate in the absence of peakedness usually takes hold at bandwidths so large that wideband fading models are called into question. Rather, ultrawideband models ought to be used.

Impact of antenna correlation on the capacity of multiantenna channels

This paper applies random matrix theory to obtain analytical characterizations of the capacity of correlated multiantenna channels. The analysis is not restricted to the popular separable correlation model, but rather it embraces a more general representation that subsumesmost of the channel models that have been treated in the literature. For arbitrary signal-to-noise ratios (SNR), the characterization is conducted in the regime of large numbers of antennas. For the low- and high-SNR regions, in turn, we uncover compact capacity expansions that are valid for arbitrary numbers of antennas and that shed insight on how antenna correlation impacts the tradeoffs between power, bandwidth and rate.

Optimum power allocation for parallel Gaussian channels with arbitrary input distributions

The mutual information of independent parallel Gaussian-noise channels is maximized, under an average power constraint, by independent Gaussian inputs whose power is allocated according to the waterfilling policy. In practice, discrete signalling constellations with limited peak-to-average ratios (m-PSK, m-QAM, etc) are used in lieu of the ideal Gaussian signals. This paper gives the power allocation policy that maximizes the mutual information over parallel channels with arbitrary input distributions. Such policy admits a graphical interpretation, referred to as mercury/waterfilling, which generalizes the waterfilling solution and allows retaining some of its intuition. The relationship between mutual information of Gaussian channels and nonlinear minimum mean-square error proves key to solving the power allocation problem.

Capacity-achieving input covariance for single-user multi-antenna channels

We characterize the capacity-achieving input covariance for multi-antenna channels known instantaneously at the receiver and in distribution at the transmitter. Our characterization, valid for arbitrary numbers of antennas, encompasses both the eigenvectors and the eigenvalues. The eigenvectors are found for zero-mean channels with arbitrary fading profiles and a wide range of correlation and keyhole structures. For the eigenvalues, in turn, we present necessary and sufficient conditions as well as an iterative algorithm that exhibits remarkable properties: universal applicability, robustness and rapid convergence. In addition, we identify channel structures for which an isotropic input achieves capacity.

Sequential estimation of multipath MIMO-OFDM channels

Wireless ���MIMO��� systems, employing multiple transmit and receive antennas, promise a significant increase of channel capacity, while orthogonal frequency-division multiplexing (OFDM) is attracting a good deal of attention due to its robustness to multipath fading. Thus, the combination of both techniques is an attractive proposition for radio transmission. The goal of this paper is the description and analysis of a new and novel pilot-aided estimator of multipath block-fading channels. Typical models leading to estimation algorithms assume the number of multipath components and delays to be constant (and often known), while their amplitudes are allowed to vary with time. Our estimator is focused instead on the more realistic assumption that the number of channel taps is also unknown and varies with time following a known probabilistic model. The estimation problem arising from these assumptions is solved using Random-Set Theory (RST), whereby one regards the multipath-channel response as a single set-valued random entity.Within this framework, Bayesian recursive equations determine the evolution with time of the channel estimator. Due to the lack of a closed form for the solution of Bayesian equations, a (Rao���Blackwellized) particle filter (RBPF) implementation ofthe channel estimator is advocated. Since the resulting estimator exhibits a complexity which grows exponentially with the number of multipath components, a simplified version is also introduced. Simulation results describing the performance of our channel estimator demonstrate its effectiveness.

Low-density parity-check codes for nonergodic block-fading channels

We design powerful low-density parity-check (LDPC) codes with iterative decoding for the block-fading channel. We first study the case of maximum-likelihood decoding, and show that the design criterion is rather straightforward. Since optimal constructions for maximum-likelihood decoding do not performwell under iterative decoding, we introduce a new family of full-diversity LDPC codes that exhibit near-outage-limit performance under iterative decoding for all block-lengths. This family competes favorably with multiplexed parallel turbo codes for nonergodic channels.

Outage probability analysis for MRC in ��-�� fading channels with co-channel interference

Exact closed-form expressions are obtained for the outage probability of maximal ratio combining in ��-�� fadingchannels with antenna correlation and co-channel interference. The scenario considered in this work assumes the joint presence of background white Gaussian noise and independent Rayleigh-faded interferers with arbitrary powers. Outage probability results are obtained through an appropriate generalization of the moment-generating function of the��-�� fading distribution, for which new closed-form expressions are provided.

Mutual information of IID complex Gaussian signals on block Rayleigh-faded channels

We present a method to compute, quickly and efficiently, the mutual information achieved by an IID (independent identically distributed) complex Gaussian signal on a block Rayleigh-faded channel without side information at the receiver. The method accommodates both scalar and MIMO (multiple-input multiple-output) settings. Operationally, this mutual information represents the highest spectral efficiency that can be attained using Gaussiancodebooks. Examples are provided that illustrate the loss in spectral efficiency caused by fast fading and how that loss is amplified when multiple transmit antennas are used. These examples are further enriched by comparisons with the channel capacity under perfect channel-state information at the receiver, and with the spectral efficiency attained by pilot-based transmission.