RICERCA ITALIANA     

Neuronal sodium, calcium and potassium channels: physiological role and channelopaties

Università degli Studi di Torino

Abstract

Voltage-gated ion channels are membrane integral proteins activated by voltage, which allow the passage of Na, Ca, K and Cl ions across the cell. All animals, including humans, take profit of the ability of ion channels to open and conduct ions through the plasma membrane in both directions, generating action potentials that sustain cell excitability. Action potentials are crucial to control vital functions such as sensory transduction, muscle contraction, synaptic plasticity, learning and memory. Usually, all these functions require a relatively low number of channels, which is in all cases surprisingly lower with respect to the number of genes available in our genoma (more than 60 genes for K channels, 10 genes for Ca and 9 for Na channels). Understanding the structure and function of each ion channel has been, and remains, the “main challenge” of neuroscientists interested on this issue.

Besides that, exits a further order of complexity associated with: 1) the functioning of the protein structures forming the pore that are often interacting with other proteins (auxiliary subunits) regulating the degree of channel expression and functioning, 2) the action of several endogenous molecules and intracellular second messengers activated by specific membrane receptors interact with ion channels, modulating their activity, and 3) the increased degree of channel expression that can be induced by neuronal growth factors and external stimuli such as hypoxia. It is obvious that a dysfunction to any of these factors and/or genetic mutations (channelopathies) that induce modifications to channel gating, or to the interaction with the auxiliary subunits and second messengers may produce severe pathologies. In this case, understanding the cause of channelopathies and consequences of genetic mutations, as well as the pathways that induce the recruitment of ion channels, may lead to important discoveries about the role of voltage-gated channels on neuronal excitability.

Here, a group of internationally qualified Italian neuroscientists has decided to participate to a joint research program with the dual purpose of studying the role of Na, Ca and K channels on neuronal functioning and the origins of several associated channelopathies. This would allow developing in the next future specific therapies for the treatment or prevention of serious neuropathologies. In this way, it will also be possible to pursue the work initiated in previous PRIN projects (1997-2007), coordinated before by prof. E. Wanke (Unit of Milano-Bicocca) and more recently by prof. E. Carbone. The last two fundings have brought excellent results: 48 scientific papers in 4 years in high rank journals and an increased image of the Italian ion channel electrophysiology at the international level certified by the increased number of invitations to national and international meetings of the project participants.

The specific competences that the five operative Units have acquired in the past ten years represent the bottom line defining the tasks of the single scientific projects. The most characterizing aspect of this project is the homogeneous expertise of the participants who have a long lasting experience in the field of ion channels and cell excitability. They are all experts of ion channels, channelopathies and electrophysiology that decided to collaborate to a joint project by exchanging their competence on different ion channel types (Na, Ca, K), neuronal preparations (cerebellum, hippocampus, neocortex, locus coeruleus, trigeminal ganglia), channelopathies (migraine, ataxia, epilepsy, Alzheimer, stress), transgenic, KI and KO mice for different channels (Cav2.1, Cav1.3, Cav3.2, Kv1.1, Kir5.1, herg1-3, Nav1.1) and most important, by using different methodologies (membrane current measurements, action potential recordings with multielectrode arrays, evoked and miniature postsynaptic currents, capacitance, amperometry, Ca imaging, confocal microscopy). If the project will be funded, it will be possible to look at the role of ion channels on neuronal functioning and on the causes of channelopathies in a more precise manner, with diversified approaches and advanced technologies already employed at the international level. As a main output of the project we expect to identify the functional role that a number of Na, Ca and K ion channels play in the generation of important neuropathologies such as migraine, epilepsy, Alzheimer and stress. <<<

Principal Investigator

Emilio Carbone Università degli Studi di TORINO

Research Objectives

This project proposal for the years 2007-2009 is the logical prolongation of that previously proposed and funded for the period (1997-2007) (coordinated before by E. Wanke and more recently by E. Carbone) with the same five research Units and a common task focused on the functional role of neuronal voltage-gated ion channels and their channelopathies. The selection of the partners has been done in the previous PRIN to better focusing on the properties of Na, Ca and K channels of the central nervous system and to allow more effective exchanges of competence on different neuronal preparations (cultured neurons, monosynaptic preparations, neuronal networks and brain slices) and modern methodologies for recording neuronal activity. The unifying element of this project is the opportunity for the five Units of effectively collaborating by joining their own experience on different neuronal preparations and a number of complex methodologies that would be nearly impossible to develop by single University groups. This is important for the final outcome of the project, which is the more deep knowledge of the role of ion channels and the origin of channelopathies at various levels of cellular complexities: single neuron, neuronal networks and entire brain.

To give an idea of the main issues that will be investigated and their extreme broadness and homogeneity, we will list here the titles of the project of the five Units, while in the graphic scheme are reported the characteristic aspects and objectives of each project: the type of channels, the channelopathies, the neurons and the methodologies used.

E. Carbone (Torino):
FUNCTIONAL ROLE OF NEURONAL L- AND T-TYPE CALCIUM CHANNELS IN THE CONTROL OF PACEMAKER ACTIVITY AND NEUROSECRETION: IMPLICATIONS IN CHANNELOPATHIES AND ALTERED CONDITIONS OF NEURONAL EXCITABILITY.

E. Wanke (Milano-Bicocca):
MOLECULAR BASES OF CHANNELOPATHIES AND FUNCTIONAL CHARACTERIZATION IN SINGLE NEURONS AND NEURONAL NETWORKS.

D. Pietrobon (Padova):
NEURONAL CALCIUM CHANNELS AND MIGRAINE

F. Tempia (Torino):
ROLE OF POTASSIUM CHANNELS IN ANIMAL MODELS OF ALZHEIMER'S DISEASE AND EPILEPSY

M. Pessia (Perugia):
PHYSIOLOGICAL ROLE OF VOLTAGE-GATED AND INWARDLY-RECTIFYING K+ CHANNELS AND THEIR IMPLICATION IN HUMAN CHANNELOPATHIES.





As it appears from the titles and from the general scheme, the project is focused on two major tasks. One concerning K channels (Kv1.1, Kv3.4, Kir5.1 and erg) in which are mainly involved the Units of Milano-Bicocca (Wanke), Torino (Tempia) and Perugia (Pessia) and the other, concerning the Ca channels involved in synaptic transmission and somatic/dendritic functions (P/Q, L, T) and the role of P/Q-type channels (Cav2.1) in hemiplegic migraine in which are implicated the Units of Torino (Carbone) and Padova (Pietrobon). A third task is also planned, about the role of Na channels in the activity of central neurons and their role in some forms of epilepsy in which the Unit of Milano-Bicocca will be involved in collaboration with a clinical group.

Although sharply focused on original topics, the five groups will nevertheless collaborate in a coordinated manner to give the necessary support to the participants to reach the final aim of each project. In this framework the three research lines on Na, Ca and K channels will proceed in parallel interacting through an effective exchange of animal models (transgenic mice, KI and KO), instrumental methodologies (MEA, measurements of evoked postsynaptic currents, Ca imaging, confocal microscopy, capacitance and amperometry), physiological preparations (cultured neurons, brain slices, intracranial injections in different brain regions) and molecular biology techniques (single-cell RT-PCR, chimeras, immunohistochemistry).

The Units involved on K channels (Milano, Perugia, Torino) will try to better understand the possible role of Kv3, Kv1.1, Kir5.1 and erg channels which control the duration of the repolarization phase, frequency of firing and interval of action potentials bursts. All these K channels appear also involved in a series of neurological diseases that include episodic ataxia, various forms of epilepsy and Alzheimer disease. The Units of Milano and Torino that have already collaborated will closely collaborate to identify the function of erg in Purkinje cells of cerebellum brain slices and hippocampal neuronal networks by using the microelectrode array technique (MEA) available in Milano. In these studies will be also used different models of Alzheimer transgenic mice. The Unit of Perugia (Pessia) will also join the project. This latter Unit, will mainly focus on the properties of Kv1.1 largely expressed in the cerebellum associated to the type-1 episodic ataxia (EA-1), in some cases associated to epileptic discharges. The Unit will use the Kv1.1 KO mice universally accepted as a good murine model of EA-1 and the Kir5.1 KO mouse which can be used as an animal model for psychiatric disorders, such as panic attach. To complete these studies a close collaboration with the Unit of Torino (Carbone) is planned to highlight the role of Kir5.1, which is expressed in chromaffin cells, in the control of cells excitability and synaptic transmission in hippocampal cells of WT and KO mice.

The two Units involved on Ca channels (Torino and Padova) will study the role of T-type (Cav3.2) and of L-type (Cav1.3) (Carbone) and P/Q-type (Cav2.1) (Pietrobon) channels on the control of neuronal excitability and neurotransmitter release in hippocampal neurons (Carbone) and in cortical and trigeminal neurons (Pietrobon), as well as the mechanisms by which mutations of P/Q-type channels associated to familial hemiplegic migraine type 1 (FHM1) cause the aura and headache symptoms of the disease (Pietrobon). Along this line, the Unit of Padova will continue the studies on KI mice carrying human FHM1 mutations and will analyze cortical excitatory and inhibitory neurotransmission and cortical network excitability to investigate the cortical mechanisms leading to increased susceptibility to cortical spreading depression (CSD, the phenomenon underlying migraine aura and a likely trigger of migraine pain) . The Unit will also focus specifically on the effects of the Cav2.1 mutation (and also of antimigraine drugs and of NO, a migraine mediator) on the trigeminal ganglion neurons involved in the development of migraine pain.

The Unit of Torino (Carbone) will study the role that L- and T-type channels exerts in the control of neuronal firing in hippocampal and chromaffin cells in two animal models: the Cav1.3 and Cav3.2 KO mice. The former mouse is deft, bradicardic and less prone to inducible stress while the latter is normally used as a model for studying forms of hyperalgesia and epilepsy. Both channels activate at relatively low voltages and are therefore implicated in the control of action potential genesis and firing behavior in a variety of neurons. The group will thus study the role of these channels in the hippocampus and chromaffin cells of WT and KO mice and will also assay the opposite effects that are expected if the two channels are up-regulated or recruited by endogenous modulators (cAMP, hypoxia) or neuronal growth factors.

All the five groups will share their expertise in the techniques they have recently developed. The group in Milano will actively collaborate with all the other partners on the extracellular recordings using MEAs in slices and cultured neuronal networks and will offer to the other units the screening &amp; site-directed-mutagenesis facility. The two groups in Torino will collaborate on setting quantitative single-cell RT-PCR (Tempia) and performing capacitative and amperometric measurements of neurotransmitter release (Carbone). <<<

First Results

As mentioned before the main output of this national research project is the identification of the functional role that a number of Na, Ca and K channels play in the generation of important neuropathologies such as migraine, epilepsy, Alzheimer, psychiatric disorders and stress. There are, however, many distinct results that each Unit is expecting to attain from their tasks and given their specific relevance to the work of each group we will list below the main outcomes expect:

TORINO (Carbone): “FUNCTIONAL ROLE OF NEURONAL L- AND T-TYPE CALCIUM CHANNELS IN THE CONTROL OF PACEMAKER ACTIVITY AND NEUROSECRETION: IMPLICATIONS IN CHANNELOPATHIES AND ALTERED CONDITIONS OF NEURONAL EXCITABILITY”

Task #1 - Role of L-type channels in pace-making cells and neurotransmitter release in hippocampal neurons and chromaffin cells under normal and altered physiological conditions.
We expect to quantify the role that the L-type channels play in the pace maker activity and hormone release in mouse chromaffin cells and hippocampal neurons. In particular, we expect to identify whether the Cav1.3 channel isoform is the one more involved and whether its up- or down regulation by external modulators will be reflected in a change of action potential activity and exocytosis. We expect also to observe the increased level of L-type channels in NGF-treated hippocampal neurons and to estimate their possible involvement in the control of neurotransmitter release at GABAergic and glutamatergic central synapses. The effects of chronic hypoxia on L-type channels are less clear and we will then search for the better conditions to induce their up-regulation.

Task #2 - Existence of distinct or converging modulatory pathways mediated by endogenous agonists on neuronal L-type channels.
Using WT, Cav1.3 and Cav1.2 KO mice we expect to discriminate whether the two types od L-type channel modulations converge on the same L-channel or they proceed in parallel on two distinct channels. The clarification of this important issue will be of great interest for the modulation of L-type channels in other tissues..

Task # 3 - T-type channels recruitment in hippocampal neurons exposed to hypoxic or other stress conditions in wild, Cav1 and Cav3 KO mice: implications on various forms of epilepsy.
Using WT and KO mice, in this task we expect to understand the role that T-type channels may exert on the action potential firing when these channel types are up-regulated by chronic hypoxia or stress-induced conditions. An increased firing activity associated with the recruitment of T-type (Cav3.2) will be relevant to the well accepted hypothesis the T-type channels play a critical role in the generation of various forms of epilepsy.



MILANO-BICOCCA (Wanke): “MOLECULAR BASES OF CHANNELOPATHIES AND FUNCTIONAL CHARACTERIZATION IN SINGLE NEURONS AND NEURONAL NETWORKS”

Task # 1.1 - Functional analysis of the missense mutation (R542Q) found in GEFS+ patients on the SCN1A gene associated with autism
The research results will clarify if the functional properties of the mutations found in patients carrying different neurological phenotypes could be clustered in similar or different classes. Indeed it is known that different types of mutations producing GEFS+ epilepsies are known to have diverse functional properties (loss-of-function, traffiking, etc). Different types of compounds acting pharmacologically will be studied for therapeutical applications.

Task # 1.2 - A human epileptogenic mutation in the KCNH7 gene (HERG3 channel) and its functional role in the CNS hetero-channels
The research results will clarify if the functional properties of the mutation (gain-of-function) that produces the absence epilepsy, putatively present in inhibitory neurons, is a property common also to other mutations known for this neurological phenotype. Different types of compounds acting pharmacologically will be studied for therapeutical applications.

Task # 1.3 - Functional properties of neuronal networks from channelopthies and degenerative diseases mice
The results of this research project will become the first to be announced and will allow to suggests the putative relationships between the observed biophysical changes and the related effects in the network activity recorded from the nervous tissue. Up to now, the changes observed in the nervous tissues were only indirectly studied, by means of behavioural methods in mice models. The applicative potentiality will be extended at pharmacological levels.

Task # 2.1 - Search for SCN1A gene mutations from a large database of the Lombardia region hospitals (about 3000 patients)
The research results will largely extend the neurological diversity and the functional properties of the identified mutations in relation to the different pathologies. This will help to clarify if the diverse biophysical properties (loss-of-function, trafficking, etc) are specifically related with various diseases. Different types of pharmacological active compounds will be studied in relation to their therapeutic use.

Task # 2.2 - Functional studies of human channelopathies models obtained by techniques of mutated gene transfer.
The results of this project should produce a strong applicative impact because the direct reconstruction, in the neuronal network, of the genetic defect will allow the use of large scale throughput screening to identify and develop new therapies and drugs.



PADOVA (Pietrobon): “NEURONAL CALCIUM CHANNELS AND MIGRAINE”

Task #1 - Analysis of cortical excitatory and inhibitory neurotransmission and cortical network excitability in WT and FHM1 knockin mice.
Concerning this task, the Unit expects to understand how the gain-of function of CaV2.1 channels produced by FHM1 mutations alters synaptic transmission and short term synaptic plasticity at excitatory and inhibitory connections between identified neurons in the cerebral cortex, and how these alterations affect the overall excitability of local cortical circuits and the balance between the total excitatory and inhibitory synaptic drive onto pyramidal neurons. We expect that this knowledge will clarify the cortical mechanisms underlying the increased susceptibility to cortical spreading depression (CSD) in FHM1. Since CSD underlies migraine aura and (according to increasing evidence) is a key primary brain dysfunction that may initiate migraine attacks, our expected findings will provide unique important insights into the controversial primary cause of migraine and may help devising new therapeutic strategies and develop much needed drugs effective in the prevention of migraine. Moreover our expected findings will establish the physiological role of CaV2.1 channels in synaptic transmission and short-tem plasticity at identified excitatory and inhibitory synapses in the somatosensory cortex as well as their physiological role in maintaining the balance between excitation and inhibition which is a key determinant of network behaviour in cortical circuits.

Task #2 - Effects of FHM1 mutations, migraine mediators and antimigraine drugs on nociceptive trigeminal ganglion neurons.
Concerning this task, the Unit expects to clarify the impact of FHM1 mutations and NO on the physiology of trigeminal ganglion (TG) neurons, in particular on the trigeminovascular neurons innervating the meninges, given their key role in the generation of migraine pain. The findings may provide insights into the neurobiology of migraine pain and the unknown mechanisms mediating the coupling between CSD and meningeal nociceptors activation. In addition we expect to improve our understanding of the mechanism of action of drugs currently used for the treatment of acute migraine attacks; this may help the development of more effective acute antimigraine drugs (20-30% of patients do not respond to the currently used drugs and headache recurrence is a common problem).



TORINO (Tempia): “ROLE OF POTASSIUM CHANNELS IN ANIMAL MODELS OF ALZHEIMER'S DISEASE AND EPILEPSY”

Task 1.1 - Investigation of alterations of spontaneous and evoked action potential discharge in neurons involved in Alzheimer’s disease.
By a thorough screening in Alzheimer’s disease models of all parameters associated with action potential discharge, the research will identify every alteration of membrane excitability due to any ionic mechanism involved. The alterations could differ across the types of neurons and between the several brain regions involved in the disease.

Task 1.2 - Identification of ionic currents and channel subunit proteins responsible for the alterations of membrane excitability and action potential discharge.
In this part of the research we expect to uncover the ionic mechanisms and the types of ionic currents responsible for each alteration discovered with the aim 1.1. These results will allow us to restrict the number of candidate channels and to identify which families and subfamilies are involved.

Task 1.3 - Gene expression analysis of the ion channels altered in Alzheimer’s disease models, by real-time RT-PCR and immunohistochemistry.
This final part of the research dealing with task # 1, will lead to the identification of the channel subunits whose expression, either at the mRNA or at the protein level, is altered in Alzheimer’s disease models.

Task 2.1 - Alterations of the expression of erg channels following experimental epileptic seizures. The results of this series of experiments will indicate for which erg channel genes the expression is up- or down-regulated following epileptic seizures. Such a result indicates a secondary involvement of one or more erg genes, which is be the basis for designing novel therapeutic strategies acting on erg currents.

Task 2.2 - Alterations of excitability following inhibition or enhancement of erg currents in acute nervous tissue slices.
Since erg channels are expressed both in excitatory and in inhibitory neurons, only experiments of this kind can provide information on their role on the overall network excitability. The results will distinguish between the roles of erg channels in different cell types in complex networks like hippocampus or thalamus-neocortex, defining the specific role of distinct neuronal populations in the spreading of excitability during seizures.

Task 2.3 - Behavioural and electrophysiological alterations of transgenic mice bearing the human epileptogenic mutation in the KCNH7 gene.
Once that the murine model of the absence epilepsy linked to the KCNH7 gene will be available, our study will define which symptoms are present by a behavioural analysis. The electrophysiological experiments will discover some of the neural mechanisms responsible for the symptoms.



PERUGIA (Pessia) “PHYSIOLOGICAL ROLE OF VOLTAGE-GATED AND INWARDLY-RECTIFYING K CHANNELS AND THEIR IMPLICATION IN HUMAN CHANNELOPATHIES”

Task #1.1 Molecular and neurophysiological mechanisms of EA1
Concerning the molecular studies of EA1, the preliminary results and those provided by the proposed experiments, which will be obtained by means of advanced cell biology and electrophysiological techniques, could demonstrate that KCNA1 mutations cause mainly loss-of-function effects. By contrast, it has been shown that EA1 mutations exert mixed effects of loss- and gain-of-function. Therefore, it is expected that this study will have a profound and general impact on EA1 and other channelopathies. On the other hand, the studies concerning the neurological mechanisms of EA1, which will be carried out by using Kv1.1KO mice, may allow the understanding of the causes of the cerebellar symptoms of EA1, such as ataxia and tremors. Moreover, it may contribute to further clarify also: i) the role of the basked cells within the cerebellar cortex circuitry; ii) the dynamics of the electrical responses of the different cerebellar cortex cell types, in normal conditions and upon Kv1.1 removal; iii) the role played by Kv1.1 channels in cerebellar short and long term plasticity events.

Task #1.2 Physiological role of the inwardly-rectifying potassium channel Kir5.1
The proposed study and the preliminary results that have been provided by using Kir5.1KO mice could demonstrate that Kir5.1 channels play an important role in modulating the potassium conductance and the excitability of LC neurons, during alkalosis and acidosis. These animals have been generated in collaboration with Dr. Tucker form the prestigious University of Oxford. Moreover, the results may shed light on the cellular mechanisms contributing to trigger panic attacks in susceptible patients upon hypercapnic acidosis. However, such potassium conductance modulation may occur also in hippocampal and cerebellar cells, where this channel type is expressed. Indeed, some experiments will be conducted in close collaboration with the other research units which may demonstrate that Kir5.1 modulates the excitability of these cells and the secretion of hormones by the Islets of Langerhans and by the adrenal glands. <<<

Timescale

24 months

National and international background

Ion channels are protein structures that play a central role in the nervous system by conditioning neuronal maturation and development of neuropathologies. In the last twenty years there has been an incredible explosion of works focused on the molecular and functional identification of different classes of Na, Ca and K channels, so that, at the moment we count more than 60 types of K channels, 10 types of Ca channels and 9 types of Na channels, with specific structural and functional properties.

Many functions are known but most remain obscure such as: 1) the molecular bases of Na, Ca and K channel gating, 2) the role of different subtypes of Na and Ca channels supporting the pace-maker current of central neurons, 3) the contribution of newly recruited Na and Ca channels in lowering the threshold of action potential firing in various forms of hyperalgesia and epilepsy, and 4) the distribution and role of different presynaptic Ca channel types responsible for vesicles release. These are only few of the many examples of unsolved problems, relative to the functions of voltage-dependent ion channels. Besides this, we are facing now a new fascinating issue of clinical interest that involves an increasing number of pathologies strictly related to the structural mutations of ion channels (channelopathies) that are identified and classified on the bases of the type of mutated channels and associated pathology. The link between “function” and “dysfunction” is so tight in this case that studying one aspect is equivalent to study the other.

1) FUNCTIONAL ROLE OF NEURONAL L- AND T-TYPE CALCIUM CHANNELS IN THE CONTROL OF PACEMAKER ACTIVITY AND NEUROSECRETION: IMPLICATIONS IN CHANNELOPATHIES AND ALTERED CONDITIONS OF NEURONAL EXCITABILITY

Most Ca channels can be effectively modulated by a variety of 2nd messengers and endogenous modulators through spatially and kinetically distinct pathways causing up- or down-regulation of Ca influx, which impairs cell activity. Of particular interest for this project is the dual modulation that L-type channels exhibit in rat chromaffin cells (RCCs) when stimulated by receptor agonists: a fast inhibition mediated by Gi-proteins and a slow potentiating action mediated by cAMP/PKA (Cesetti et al, 2003). A possibility is that the two modulatory mechanisms converge on the same L-type channel isoform (Cav1.2), but it can also be that each pathway targets one of the two L-type channels expressed by RCCs (Cav1.2 &amp; Cav1.3). The existence of these two isoforms in central neurons and the key role that neuronal L-type channels play in the pathogenesis of memory, depression, Alzheimer and Parkinson diseases (Chan et al, 2007) makes extremely interesting to investigate the existence of these pathways also in hippocampal neurons.

Ca channels can also be effectively recruited by short and long term treatments that involve neurotrophic factors, intracellular 2nd messengers, neurotransmitters and endogenous modulators which generate transcriptional signals to the nucleus and stimulate Ca channel synthesis and incorporation in the membrane. Recruitment and modulation of Ca channels is an important key step in neuronal functioning which can lead to drastic changes to cell functioning (Carbone et al, 2006)]. We recently showed that long-term treatment of hippocampal neurons with NGF causes a selective up-regulation of L-type channels which accelerates the maturation process of these neurons (Baldelli et al., 2005). In parallel to this, we also showed that single applications of cAMP or beta-adrenergic stimulation can effectively recruit alpha1H T-type channels in RCCs after 3-4 days of cell incubation. Recruitment of T-type channels triggers a significant “low-threshold” exocytosis with Ca-efficiency similar to that of L-type channels. Recruitment of T-type channels occurs also during chronic hypoxia conditions via hypoxia-inducible factors (Carabelli et al, 2007). This suggests that stressful conditions stimulate the recruitment of T-type channels in RCCs, possibly through a common transcriptional mechanism leading to the new synthesis of functioning T-type channels.

Given the strategic importance of L- and T-type channels in hippocampal neurons and chromaffin cells functioning the research activity of this project will focus on three tasks: 1) the role of L-type channels in pace-making cells and neurotransmitter release in hippocampal neurons and chromaffin cells 2) the existence of distinct or converging modulatory pathways mediated by endogenous agonists on neuronal L-type channels, 3) the recruitment of T-type channels in hippocampal neurons exposed to hypoxic or other stress conditions in wild and Cav3 KO mice.


2) MOLECULAR BASES OF CHANNELOPATHIES AND FUNCTIONAL CHARACTERIZATION IN SINGLE NEURONS AND NEURONAL NETWORKS.

The 2007 project involves not only the ending of previous work (grant PRIN-2005) on the SCN1A gene (Nav1.1) and KCNH7 gene (Kv11.3; herg3), but also the beginning of a new series of studies with a novel electrophysiological technique, the multi-electrode recording (MEA). The preliminary results of this research, still unpublished, are illustrated below:

2.1 – Evidence that a SCN1A gene mutation associated with autism is also present in a GEFS+ epilepsy. During a recent gene screening we discovered that a mutation was present in a family classified as GEFS+ epilepsy, but the same mutation has been described previously as associated with autism (Weiss et al, 2003). The mutation is an aminoacid change R542Q located in the cytoplasmic region linking domains I and II of the protein. This mutation results in a well conserved aminoacid among the most common isoforms in the CNS of mammals. Moreover, the eight subsequent aminoacids have been considered as a consensus sequence for a site that was predicted to be able to phosphorilate the channel itself. Since it is known that the channel can be modulated by tyrosine phosphorilations, the mutation could alter the activity of the channel, suggesting that it functional properties of this mutated channel, never studied before, merit a study.

2.2 – Update of the research project on mutations on ion channels of the herg K-type
The superfamily of the ERG (EAG-related-gene) K+ ion channels is composed, in mammals, by three members. All the genes are strongly expressed also in the nervous system. The ERG currents are suitable to control various physiological functions well studied in our laboratory (Chiesa et al., 1997) and have been also suggested that it could be linked to tumour proliferation. A HERG3 mutation has been detected in a patient with an epilepsy with absence characterized by prof. Bianchi (Arezzo) and Zara (Gaslini, Genova). Its expression in HEK292 cells obtained and characterized in our laboratory consists in a left shift of the voltage-dependent activation with the consequence of a “gain-of-function” mutation.

2.3 – Properties of advanced electrophysiological techniques
The multielectrode recording (MEA) is a novel non-invasive technique that allows the simultaneous analysis of the electrical activity of hundreds neurons by extracellular capture of single action potentials. The use of the MEA allows studies of the spatio-temporal activity both in structured slices or organotipic cultures and in unstructured (Tu et al, 2005) neuronal networks. We have evidence that it is possible to change the homeostasis of the extracellular GABA which is finely controlled by the released GABA and the GABA uptaken by the GAT1 transporter. By adding increasing concentrations of the competitive inhibitor of GAT1 (SKF89976A), it is possible to observe a net decrease of the activity with the same IC50. Moreover, it is known that carbenexolone (CBX) is able to block the electrical synapses (gap junctions) between inhibitory interneurons. Also in this case, it is possible to demonstrate the functional role of the gap junctions present in the networks and that increasing concentrations of the drug are able to produce opposite effects in different clusters of the neurons firing in the network. To illustrate the complete development of the GABAergic system and the presence of the pacemaker channels in the neurons, we observed the effects of gabazine (GBZ, 20µM, a potent GABA inhibitor) which potently disinhibits the network activity. On the contrary, the drug ZD7288 (a blocker of the pacemaker, HCN channel), which sustains the depolarizing cationic current was able to completely inhibit the network activity suggesting its crucial contribution to the network activity.


3) NEURONAL CALCIUM CHANNELS AND MIGRAINE

Among the different neuronal voltage-gated Ca channels, the P/Q-type Ca (CaV2.1) channels have a dominant role in controlling fast synaptic transmission particularly at central excitatory synapses (Pietrobon, 2005). Mutations in the gene encoding the pore-forming subunit of these channels cause a number of autosomal dominant neurological diseases, that include familial hemiplegic migraine type 1 (FHM1), a rare subtype of migraine with aura. Migraine is one of the most prevalent neurological diseases, affecting more than 10 % of the population, and ranked by WHO as one of the 20 most disabling diseases. There is good evidence that i) migraine visual aura is the result of cortical spreading depression (CSD: a wave of neuronal depolarisation that spreads slowly across the cerebral cortex, followed by neural suppression; ii) the development of migraine pain depends on the activation and sensitization of trigeminal nociceptive sensory fibers innervating the meninges; iii) the primary cause of migraine lies in the brain. However, incompletely understood issues are the nature and mechanisms of the primary brain dysfunction causing migraine and the main site and basic mechanisms of generation and maintenance of migraine pain. Recent animal studies support the idea that CSD is a key primary brain dysfunction that may initiate migraine attacks (van den Maagdenberg et al, 2004)].

We have shown that FHM1 mutations shift to lower voltages the activation of human recombinant CaV2.1 channels and increase the single channel open probability and Ca influx in a broad voltage range, and have revealed a similar increase in neuronal P/Q-type Ca current density in cerebellar and cortical neurons of KI mice carrying FHM1 mutations, leading to the conclusion that FHM1 mutations produce gain-of-function of human and mouse CaV2.1 channels. Recently we have shown that gain-of-function of presynaptic P/Q channels results in increased probability of glutamate release at cortical synapses of KI mice (Conti et al, 2007)]. Moreover, we have shown that FHM1 KI mice have a lower threshold for CSD initiation and an increased velocity of CSD propagation, indicating an increased susceptibility to CSD.

The consequences of FHM1 mutations on trigeminovascular nociceptive pathways remain largely unexplored. It has been shown that P/Q channels control both CGRP release from perivascular terminals of meningeal nociceptive afferents and neurogenic vasodilation in vivo [38,40], and we have shown that they control somatic release from trigeminal ganglion (TG) neurons and contribute to the long duration of their action potential (unpublished observations). We have distinguished three different types of TG neurons on the basis of firing, capsaicin-sensitivity and expression of low-voltage-activated Ca channels and have revealed different gain-of function of the P/Q Ca current in different TG neurons of FHM1 KI mice (Pietrobon et al, 2007).


4) ROLE OF POTASSIUM CHANNELS IN ANIMAL MODELS OF ALZHEIMER'S DISEASE AND EPILEPSY

The first part of the project (task #1) is devoted to the study of alterations of membrane excitability in models of Alzheimer’s disease, with a special attention to potassium channels, which are the main mechanism underlying this function. The experiments of this part of the research are based on findings funded by the previous COFIN-PRIN-2005 project, in the aspect regarding Kv3 currents. More specifically, the Kv3.4 subunit is over-expressed in patients with Alzheimer’s disease (Angulo et al, 2004), suggesting an involvement in such disorder. This conclusion is confirmed by recent in vitro data implying the Kv3.4 subunit in apoptosis provoked by the beta-amyloid peptide. Although these studies clearly indicate that the beta-amyloid peptide induces an increase of the Kv3.4 current, involved in the neurodegenerative process, the functional consequences of such increase on membrane potential and excitability have not been investigated. For these reasons, the study of potassium channels of the Kv3 subfamily will continue in the PRIN-2007 project, in relation to their role in Alzheimer’s disease which was not an aim of the previous projects. However, the issue of the alterations of membrane excitability in models of Alzheimer’s disease will be extensively investigated, including all other families and subfamilies of potassium channels. At present, there are a few reports on this subject, each indicating the alteration of a different type of potassium current: Kv3.4, Kir2.2, Ca dependent K channels, A-type K currents or potassium currents responsible for the action potential after-hyperpolarisation.

The second part of the project (task # 2) is aimed at finding the involvement of erg potassium channels in epilepsy. In the previous projects (COFIN-PRIN-2003 and 2005) we have shown that erg currents reduce the excitability of a neuron of the central nervous system and that the pattern of expression of erg subunits is in line with a role in epilepsy (Guasti et al, 2005).


5) PHYSIOLOGICAL ROLE OF VOLTAGE-GATED AND INWARDLY-RECTIFYING K+ CHANNELS AND THEIR IMPLICATION IN HUMAN CHANNELOPATHIES

Episodic ataxia type-1 (EA1) is an autosomal dominant neurological disorder affecting both the central and peripheral nervous systems. EA1 is characterized by myokymia and by episodic attacks of ataxia with loss of balance, which can be life threatening. Affected individuals bear one of a number of point mutations in the Shaker-related, V-dependent K channel gene KCNA1 (hKv 1.1) on chromosome 12p13. EA1 channels display altered gating (D’Adamo et al, 1998). Recently, in collaboration with neurologists and geneticists of the University of Ferrara and, with the support of our PRIN2005, we have identified a new mutation in a Sicilian family affected by EA1 and epilepsy. The functional characterization study revealed a complete loss-of-function of the mutated homomeric channel and altered Kv1.1/Kv1.2 channel gating (Imbrici et al, 2003). Since several mechanisms involved in EA1 remain elusive, we propose to address some of the unresolved questions such as: do mutations associated with EA1 really cause a mixed loss- and gain-of-function of the channel? (task # 1); Which are the altered mechanisms occurring at the central circuits that result in ataxia, tremors and epilepsy? (task # 2).

Inwardly-rectifying K channels are found in almost every cell type where they function as essential regulators of K fluxes across membranes (Pessia, 2004). The physiological role of Kir5.1 channels remains unknown. Kir 5.1 does not produce functional K channel activity when expressed by itself. Instead it appears to selectively coassemble with Kir4.1 and Kir4.2. The high sensitivity of heteromeric Kir4.x/Kir5.1 channels to protons, together with their tissue distribution, suggest that they play a key role as regulators of potassium flux during alkalosis and acidosis. This regulation may occur either in the CNS (Locus coeruleus, etc.) or in other tissues such as renal tubules, pancreas acinar cells, islets of Langerhans, adrenal glands. To establish the physiological role of this channel type we have recently generated Kir5.1 knockout mice, in collaboration with Dr. S. Tucker (Oxford University), that are currently utilized by our research unit (task #3). <<<