Lectures will take place in the Academy of Science building (see how to find it in section locations)

Lecture Program

Thursday October, 14th.

Introductory remarks


Lev Magazanik, Yuri Natochin

Russian Academy of Sciences & Sechenov Institute


David Brown

Physiological Society


Theme 1: transmembrane channels


David Brown
Department of Pharmacology, University College London, London, WC1E6BT, UK


KCNQ (Kv7) channels: a family of voltage-gated potassium channels

KCNQ (Kv7) channels are a small family of voltage-gate potassium (K+) channel subunits, comprising 5 members (KCNQ1-5). Remarkably, spontaneous mutations in 4 of these give rise to known human genetic diseases (see ref.1). Q1 is present in the heart and in some epithelia, and forms channels in combination with members of the KCNE auxiliary subunits; mutations in Q1 (or KCNE1) cause some forms of the cardiac 'long-QT' syndrome. Q2-Q5 are restricted to the nervous system - Q4 primarily to the auditory and vestibular systems, whereas Q2, 3 and 5 are more widely distributed and constitute subunits of the 'M-channel', a subthreshold K+ channel that regulates neuronal excitability. Although the individual subunits can form functional homomeric channels, the native M-channels are probably heteromers of Q3/2 and/or Q3/5. Minor mutations in Q2 or 3 cause a form of epilepsy called Benign Familial Neonatal C onvulsions (BFNC); mutations in Q4 cause a form of progressive hereditary deafness. Architecturally, KCNQ channels are similar to other Kv channels, but are distinguished by their long and variable C-terminus. They also have a unique pharmacology: they are resistant to conventional K+ channel blockers (except Q2, which is blocked by TEA), but blocked by the DuPont-Merck compounds linopirdine and XE991; and their activity is enhanced (through an activation voltage-shift) by an anticonvulsant, retigabine. They are also regulated by membrane phosphatidyl-inositol-4,5-bisphosphate (PIP2) and variably by intracellular Ca2+ (via channel-associated calmodulin) and by protein kinase C (PKC), and are inhibited by transmitters acting on receptors coupled to the G proteins Gq and G11 (ref.2), providing a mechanism whereby such transmitters can increase neuronal excitability (ref.3). Recently they have been implicated in the regulation of nociceptive transmission (ref.4).

  1. Jentsch, T.J. (2000). Neuronal KCNQ potassium channels: physiology and role in disease. Nat. Res. Neurosci., 1, 21-30 [PDF]
  2. Selyanko, A.A., Hadley, J.K., Wood, I.C., Abogadie, F.C., Jentsch, T.J. & Brown, D.A. (2000). Inhibition of KCNQ1-4 channels expressed in mammalian cells via M1 muscarinic acetylcholine receptors. J.Physiol., 522, 349-355. [PDF]
  3. Brown, D.A. (1983). Slow cholinergic excitation - a mechanism for increasing neuronal excitability. Trends in Neurosci., 6, 302-307.
  4. Passmore, G.M., Selyanko, A.A., Mistry, M., Al-Qatari, M., Marsh, S.J., Matthews, E.A., Dickenson, A.H., Brown, T.A., Burbidge, S.A., Main, M. & Brown, D.A. (2003). KCNQ/M currentsin sensory neurons: significance for pain therapy. J. Neurosci., 23, 7227-7236. [PDF]


Coffee break


David Marples
School of Biomedical Sciences, University of Leeds


Aquaporin water channels in health and disease

The aquaporins are a recently discovered, but ancient and widespread, family of proteins, expressed in plants, animals, and prokaryotes, but in many cases their functional importance remains to be discovered. They act as water channels, and some also allow the passage of glycerol. Eleven AQPs have been identified in man, some of which are widespread in the body while others have specific roles.
The best characterised is AQP1, which is abundantly expressed in red cells, making it easy to purify, and its structure has now been determined at the atomic level. The proteins exist in the form of tetramers, but in contrast to most ion channels, each subunit of the tetramer acts as a channel. Each monomer has six transmembrane helices arranged as a "barrel", with cytosolic N and C termini. The first intracellular and last extracellular loops, which contain NPA domains, fold into the middle, forming the water channel. The NPA domains come together to provide a "selectivity filter", which allows the rapid (109 molecules/sec) but selective movement of water: even electrons in the form of H3O+ are unable to pass.
While some aquaporins, such as AQP1 and AQP4, appear to be constitutively inserted into the plasma membrane, others such as AQP2 are regulated by shuttling to and from a target membrane. There is limited evidence for regulation of aquaporins by gating. Longer term regulation occurs by changes in transcription and/or translation.
Mutations in AQP2 lead to nephrogenic diabetes insipidus, and AQP0 (MIP) mutations cause cataracts. In contrast, AQP1-null humans appear phenotypically normal, while AQP4 knockout mice are well and have increased tolerance to strokes! It is clear that much work remains to be done on the physiology and pathophysiology of aquaporins.

  1. Agre P. & Kozono D. (2003) FEBS letters 555 72-78. [PDF]
  2. Nielsen S., et al. (2002) Physiol. Rev. 82, 205 - 244. [PDF]
  3. Maurel C. & Chrispeels M. (2001) Plant Physiol. 125, 135 - 138. [PDF]
  4. Johansson I., et al. (2000) Biochim. Biophys. Acta 1465, 324 - 342 [PDF]


Denis Tikhonov
Sechenov Inst. of Evolutionary Physiology and Biochemistry, St.Petersburg, Russia


Ion channels structure and functions: experiments in silico

It is commonly accepted that channel blockers of the nicotinic acetylcholine receptors (nAChR) bind to the hydrophobic Leucine ring and to polar Serine and Threonine rings in the pore-lining M2-segments. However, structure-activity data in the series of philantotoxin (PhTX) derivatives apparently disagree with this binding scheme. Replacement of two secondary amino groups in PhTX-343 by methelene groups (PhTX-(12)) lead to prominent increase of blocking activity. This paradox stimulated a series of experimental and modeling studies. It was demonstrated that unlike PhTX-343, PhTX-(12) causes voltage-independent block [1]. Calculations of the optimal 3D structures showed that closing of intramolecular H-bonds result in markedly different shape of the molecules. Docking of the toxins to the nAChR channel model revealed corresponding difference in the optimal binding modes. PhTX-343 binds deep in the pore near the Serine ring where classical open channel blockers of nAChR bind. In contrast, PhTX-(12), which has a single charged amino group is unable to reach deeply located rings because of steric restrictions [2].
Crystallographic studies of K channels in the closed and open states suggest that conserved Gly in the inner helices serves as a gating hinge during channel activation. However, some P-loop channels have larger residues in the corresponding position. This raises questions about the mechanism of conformational changes upon channel gating. The problem cannot be solved by X-ray structures, which are static pictures. Opening and closing of KcsA by were simulated by constraining C-ends of the inner helices at a gradually changing distance from the pore axis without restraining mobility of the helices along the axis. The channel-opening and channel-closing trajectories arrived to the structures in which the backbone geometry was close to that seen in X-ray structures. In the channel-opening trajectory, the constraints-induced lateral forces caused kinks at midpoints of the inner helices but did not destroy interdomain contacts, the pore helices, and the selectivity filter. The simulated activation of the Gly99Ala mutant yielded essentially similar results. Analysis of energetics shows that the N-terminal parts of the inner helices form strong attractive contacts with the pore helices and the outer helices. The lateral forces induce kinks at the position where the helix-breaking torque is maximal and the intersegment contacts vanish. This mechanism may be conserved in different P-loop channels [3].

  1. Brier T.J., Mellor I.R., Tikhonov D.B., Neagoe I., Shao Z., Brierley M.J., Stromgaard K., Jaroszewski J.W., Krogsgaard-Larsen P., Usherwood P.N. 2003. Contrasting actions of philanthotoxin-343 and philanthotoxin-(12) on human muscle nicotinic acetylcholine receptors. Mol Pharmacol. 64:954-964.
  2. Tikhonov D.B., Mellor I.R., Usherwood P.N. 2004. Modeling noncompetitive antagonism of a nicotinic acetylcholine receptor. Biophys J. 87:159-170.
  3. Tikhonov D.B., Zhorov B.S. 2004. In Silico Activation of KcsA K Channel by Lateral Forces Applied to the C-Termini of Inner Helices. Biophys J. 87 (In press)



Friday October, 15th.

Theme 2: Membrane Transporters


Clive Ellory
University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT, UK


Cation chloride co-transporters

There are several important members of the cation chloride cotransporter (CCC) family which are involved in cell volume regulation, cell growth, and proliferation. As well as the Na-K-2Cl (NKCC) cotransporter which is important in the ascending limb of the Loop of Henle, there is the Na-Cl cotransporter in the early distal tubule which is involved in salt reabsorption, and four isoforms of the K-Cl- cotransporter, KCC 1-4. KCC 1 and 3 were originally shown to be activated by dephosphorylation and to perform KCl efflux in swollen cells for regulatory volume decrease (RVD). I n many cells NKCC is activated by phosphorylation and increases cell volume (RVI), whist KCCs respond to dephosphorylation to perform RVD. In sickle red blood cells, KCC is abnormally active, and is responsible for cellular dehydration and eventual sickling.
KCC2 is neuron-specific, and critical for the maturation of inhibitory GABA responses in the nervous system by the control of intracellular chloride concentration. In cervical cancer cells, KCC1,3 & 4 expression is regulated during the cell cycle, and is increased compared to normal cells. The KCC activity correlates with the proliferation, growth and spread of the cancer, reflecting the role of these transporters in volume regulation during cell growth and division. The KCC activity is also stimulated by certain growth factors, particularly IGF-1, acting via kinases (P13K/Akt; Erk1/2 MAPK) to promote gene transcription and synthesis of KCC. Because transport is coupled and electroneutral, KCC activity cannot be measured electrophysiologically, but will involve radioisotope and optical techniques.

  1. Shen MR, Chou CY, Hsu KF, Hsu YM, Chiu WY, Tang MJ, Alper SL, Ellory JC. (2003). KCl cotransport is an important modulator of human cervical cancer growth and invasion. J.Biol. Chem. 278: 39941-50 [PDF]
  2. Shen MR, Chou CY, Hsu KF, Liu HS, Dunham PB, Holtzman EJ, Ellory JC (2001). The KCL cotransporter isoform KCC3 can play an important role in cell growth regulation Proc. Natl. Acad. Sci. USA 98: 14714-9 [PDF]


Florian Lang, Ekaterina Shumilina, Nikita Gamper, Alexeij Vereninov, Monica Palmada, Christoph Bohmer, Amanda Wyatt, Susanne Berchtoldt, Siegfried Waldegger
University of Tubingen


Diverse functions of the transport regulating kinase SGK1

The Serum and Glucocorticoid inducible Kinase SGK1 stimulates a variety of transport molecules (channels, pumps, carriers) including the Na+ channel ENaC and SCN5A, the K+ channels ROMK1, Kv1.3, and KCNE1/KCNQ1, the Ca2+ channel TRPV5 and the Cl- channels CFTR, ClC2 and ClC-Ka, the Na+/K+ ATPase, the Na+/H+ exchanger NHE3, the glucose carrier SGLT1, the phosphate carrier NaPiIIb, the amino acid transporters and SN1, EAAT1 and EAAT3 as well as the citrate transporter NaDC. The functions are partially shared by the isoforms SGK2 and SGK3. The kinases regulate channel/transporter abundance in the plasma membrane in part by inhibition of the ubiquitin ligase Nedd4-2 and in part by interaction with trafficking molecules such as the Na+/H+ exchanger regulating factor NHERF2. An in vivo role of SGK1 mediated transport regulation is illustrated by the sgk1 knockout mouse (sgk1-/-). In the absence of challenges the mouse appears to be seemingly normal but appropriate experimental conditions unmask the impaired ability of sgk1-/- mice to adequately regulate transport. The sgk1-/- mouse does not sufficiently reduce renal Na+ output and is unable to maintain blood pressure during dietary salt restriction, its ability to excrete a potassium load is decreased and it does not respond with enhanced intestinal glucose absorption following treatment with glucocorticoids. The functional significance of SGK1 is further documented by enhanced blood pressure, shortened cardiac action potential and increased body mass index of individuals carrying certain polymorphisms in the SGK1 gene. Those individuals may be particularly prone to suffer from metabolic syndrome, a disorder characterized by obesity, hypertension, and enhanced risk to develop diabetes mellitus and cardiovascular disease. Thus, regulation of transport by the SGKs participates in diverse physiological functions and pathophysiological conditions.

  1. Busjahn A, Aydin A, Uhlmann R, Feng Y, Luft FC, Lang F. Serum- and glucocorticoid-regulated kinase (SGK1) gene and blood pressure. Hypertension 40(3): 256-260, 2002
  2. Dieter M, Palmada M, Rajamanickam J, Aydin A, Busjahn A, Boehmer C, Luft FC, Lang F. Regulation of glucose transporter SGLT1 by ubiquitin ligase Nedd4-2 and kinases SGK1, SGK3, and PKB. Obes Res. 2004 May;12(5):862-70.
  3. Firestone GL, Giampaolo JR, O'Keeffe BA. Stimulus-dependent regulation of serum and glucocorticoid inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity. Cell Physiol Biochem. 2003;13(1):1-12
  4. Lang F, Cohen P. Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms. Science STKE (13): (108):RE17, 2001
  5. Lang F, Henke G, Embark HM, Waldegger S, Palmada M, Bohmer C, Vallon V. Regulation of channels by the serum and glucocorticoid-inducible kinase - implications for transport, excitability and cell proliferation. Cell Physiol Biochem. 2003;13(1):41-50.
  6. Pearce D. SGK1 regulation of epithelial sodium transport. Cell Physiol Biochem. 2003;13(1):13-20.
  7. Verrey F, Loffing J, Zecevic M, Heitzmann D, Staub O. SGK1: aldosterone-induced relay of Na+ transport regulation in distal kidney nephron cells. Cell Physiol Biochem. 2003;13(1):21-8.
  8. Wulff P, Vallon V, Huang DY, Volkl H, Yu F, Richter K, Jansen M, Schlunz M, Klingel K, Loffing J, Kauselmann G, Bosl MR, Lang F, Kuhl D. Impaired renal Na+ retention in the sgk1-knockout mouse. J Clin Invest 110(9):1263-1268, 2002


Coffee break


Richard Boyd


Cell and molecular studies on PepT1: a transporter with important roles in nutrition, drug delivery and the brain

PepT1 is an epithelial peptide transporter found in the brush border membrane, particularly of the small intestine and proximal renal tubule. It was cloned by expression nearly 10 years ago and has properties that make it particularly interesting, both physiologically and for clinical medicine. It transports as naturally occurring substrates all di- (202=400) and tri- (203=8000) peptides. Additionally, it transports a wide variety of peptide-like drugs such as the antibiotics penicillin. The mechanism of transport involves secondary active transport coupled to the H+ electrochemical gradient, rather than the sodium gradient (the more usual coupling process in vertebrate cells).
In this talk I will review recent findings concerning the substrate specificity, the electrogenicity and the proton coupling stoichiometry. I will also discuss recent molecular observations that show how the coupling cycle is interrupted by site-specific mutations within the protein; and how co-injection experiments have revealed that the transporter functions as a multimer, probably involving as yet unidentified additional protein accessory proteins.

  1. Meredith D, Boyd CAR (2000). Structure and function of eukaryotic peptide transporters. Cell Mol Life Sci. 57:754-78. [PDF]
  2. Meredith D (2004). Site-directed mutation of arginine 282 to glutamate uncouples the movement of peptides and protons by the rabbit proton-peptide cotransporter PepT1. J. Biol. Chem. 279:15795-8. [PDF]


Anna Bogdanova


Na/K pump function in neuronal primary culture: redox- and oxygen-sensitivity



Saturday October, 16th.

Theme 3: Cation Channels




David Beech
School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK


Mammalian TRPC cationic channels: activation, regulation and physiological functions

For many years, studies of smooth muscle and other cell types have shown pharmacological agents and physiological agonists activate a range of non-selective cationic channels referred to variously as store-, receptor- or second messenger- operated, or capacitative calcium-entry channels (McFadzean & Gibson 2002). The molecular basis of these channels has been uncertain, but an ever-strengthening hypothesis is that they are encoded by mammalian homologues of Transient Receptor Potential (TRP) - a Drosophila gene with a role in the light response of the fly's photoreceptor. In excessive of twenty mammalian TRP homologues are known and they have a range of very interesting properties (Clapham 2003). For example, there are studies suggesting TRPC1 is a functional component of store-operated channels in diverse cell types (Beech et al 2003). We have developed specific antibody to TRPC1 and shown TRPC1 is expressed in smooth muscle. The antibody is functional - partially inhibiting store-operated calcium entry and endothelin-evoked contraction in arteries (Xu & Beech 2001; Bergdahl et al 2003). However, heterologous o ver-expression of TRPC1 alone does not produce functional channels, leading us to search for other TRPCs that could be partners of TRPC1. Several TRPCs are expressed and a focus on TRPC5 has revealed it is a glycosylated protein near the plasma membrane of smooth muscle cells. Antibody targeted to TRPC5 blocks function of over-expressed TRPC5 as well as the store-operated signal in arteries. Heterologous expression studies show TRPC5 functions as a homo- and hetero-meric complex and is activated by a range of different signals - a promiscuity of gating that is an emerging feature of TRP channels. Therefore, mammalian TRPC proteins may be the molecular basis of several non-selective cationic channels in smooth muscle and the proteins display unusual and striking activation characteristics.
Supported by the Wellcome Trust and British Heart Foundation.

  1. McFadzean & Gibson 2002, Br J Pharmacol 135, 1-13. [PDF]
  2. Clapham, 2003, Nature 426, 517-524. [PDF]
  3. Beech et al 2003, Cell Calcium 33, 433-440. [PDF]
  4. Xu & Beech 2001, Circulation Research 88, 84-87. [PDF]
  5. Bergdahl et al 2003, Circulation Research 93, 839-847. [PDF]


Stuart Bevan
Novartis Institutes for Biomedical Research, London, UK


TRP channels and thermosensation by sensory nerves

The discovery that several members of the Transient Receptor Protein (TRP ) channel family are thermosensitive with threshold temperatures for activation spanning the physiological (and environmental range) has transformed our understanding of temperature sensation. This talk will review the current knowledge of TRP channel thermosensitivity and describe our discovery of TRP channels sensitive to warm and cold temperatures. TRPV1 (formerly known as VR1 or the capsaicin receptor), which is expressed predominantly in small diameter primary afferent neurons, is activated at temperatures ? 42oC and is responsible for sensing noxious heat. TRPV1 activity is modulated by a range of mediators generated during inflammation. Some of these mediators directly activate TRPV1 while others sensitize the channel via intracellular second messenger pathways. For this reason TRPV1 appears to have an important role in the sensation of pain associated with inflammation.
We have used a bioinformatics approach to search for new members of the TRP channel family. From this search we discovered and characterized other thermosensitive channels. TRPV3 is a warm sensitive channel that is activated with a threshold temperature of ~30oC. The expression pattern of TRPV3 in skin cells and in a subset of sensory neurons is well suited to a role as a warm sensor. TRPM8 is expressed in a sub-population of sensory neurons distinct from TRPV1 expressing neurons. TRPM8 is activated at cool temperatures (threshold ~20oC) and also by the cooling agents menthol and icilin. These agents act to raise the threshold for temperature activation so that 'cool' fibres are activated at normal body temperature. Finally TRPA1 which expressed in neurons expressing TRPV1 is activated by cold temperatures, as well as pungent agents such as mustard oil. References

  1. Peier AM, Reeve AJ, Andersson DA, Moqrich A, Earley TJ, Hergarden AC, Story GM, Colley S, Hogenesch JB, McIntyre P, Bevan S, Patapoutian A. (2002) A heat-sensitive TRP channel expressed in keratinocytes. Science 296: 2046-2049. [PDF]
  2. Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story GM, Earley TJ, Dragoni I, McIntyre P, Bevan S, Patapoutian A. (2002) A TRP channel that senses cold stimuli and menthol. Cell 108:705-715. [PDF]
  3. Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A.(2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell: 112 819-829. [PDF]


Coffee break


Alan North
Faculty of Life Sciences, University of Manchester, England, UK


P2X receptor channels

P2X receptors mediate excitatory postsynaptic potentials in some parts of the central and peripheral nervous systems. They are also involved in the initiation of primary afferent signals, being released from epithelia in the walls of viscera. The receptors form as multimers of several (perhaps three) subunits which can be the same or different. There are seven P2X subunits in mammals, and the P2X3 subunit is found almost exclusively in a subset of primary afferent nerves. P2X receptor subunits are unrelated in amino acid sequence to other ion channel or nucleotide binding proteins. We have sought to determine how the proteins operate as ion channels by the substituted cysteine accessibility method. T his involves mutation of the cDNA so as to replace individual amino acids by cysteine; the cDNA is then expressed in human embryonic kidney cells from which whole-cell recordings are made. The accessibility of the cysteine can then be examined by applying methanethiosulphonate (MTS) compounds. By using MTS compounds of different charges and size, a variety of side-chains can be attached to the channel protein; for example, methyl (-S-S-CH3), sulfonatoethyl (-S-S-CH2-CH2-SO3-), or ethylammonium (-S-S-CH2-CH2-NH4+). The effects of membrane current evoked by ATP are then measured. These experiment have allowed us to identify parts of the protein involved in ion permeation and ATP binding, as well as residues that are sufficiently close to form disulfide bridges. This indicates that the P2X2/3 heteromeric channel that predominates on pain-sensing primary afferent neurons is probably composed of two P2X3 subunits and one P2X2 subunits. Mutations in putative ATP binding sites on the P2X2 and the P2X3 subunits in the heteromeric channel strongly indicates that the binding site for a single ATP molecule is contributed by amino acid residues from two subunits. This finding may be useful for the further development of drugs that block such heteromeric receptors, and which are valuable in for the relief of neuropathic and inflammatory pain.

  1. North, R.A. The P2X3 subunit: a molecular target in pain therapeutics. Curr. Opin. Invest. Drugs 4: 833-840, 2003. [PDF]
  2. Jiang, L.-H., Kim, M., Spelta, V., Bo, X., Surprenant, A. & North, R.A. Subunit arrangement in P2X receptors. J. Neurosci. 23: 8903-8910, 2003.
  3. North, R.A. The molecular physiology of P2X receptors. Physiol. Rev. 82: 1013-1067, 2002. [PDF]


Stuart Cull-Candy
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK


Glutamate receptor subunits: the key to central synaptic transmission

We are interested in how nerve impulses are transmitted between neurons in the brain. At excitatory synapses, glutamate diffusing across the cleft binds to specific in postsynaptic receptor molecules, that contain an integral ion channel. At many excitatory synapses, the transmitter released into the synaptic cleft activates a mixture of NMDA- and AMPA receptors. These can be composed of a variety of different subunits that critically determine the functional properties of individual receptors and hence of central synaptic transmission.
You will recall that there are at least four distinct AMPAR subunits (GluR1-4), and at least five main NMDA receptor subunits. The GluR 2 AMPAR subunit is subject to RNA editing. The presence of an edited subunit in the receptor assembly has a marked effect on the receptor channel properties, the most notable being a dramatic reduction in Ca2+-permeability of GluR2 containing receptors. The NMDA receptor subunits consist of two separate families: NR1, which consists of a single subunit (with a eight splice variants), and the NR2 family which consists of four members - 2A, -2B, and -2C and 2D. A third family of NMDAR subunits has been identified, but these have not been widely studied.
I will focus on the roles played by certain AMPA- and NMDA receptor subunits in synaptic transmission. First I will describe evidence for the presence on an activity dependent switch in the subunit composition and Ca2+-permeability of synaptic AMPARs in cerebellar stellate cells (Liu & Cull-Candy, 2000; 2002). Second I'll describe evidence for the presence of two functionally distinct types of NMDA channels in cerebellar cells (Cull-Candy et al 2000)). Third, I'll address the issue of which of the functionally distinct classes of NMDAR-channel are activated during synaptic transmission (Cathala et al. 2002; Brickley et al 2003).

  1. Brickley, S. G., Misra, C., Mok, M-.H. S , Mishina, M & Cull-Candy, S,G. (2003) NR2B and NR2D subunits co-assemble in cerebellar Golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites Journal of Neuroscience 23, 4958-4966 [PDF]
  2. Cull-Candy, S. G., Brickley, S.G. & Farrant, M. (2001) NMDA receptor subunits: diversity, development and disease. Current Opinions in Neurobiology 11, 327-335. [PDF]
  3. Liu, S-Q. J. & Cull-Candy, S. G. (2000) Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype. Nature 405, 454-458. [PDF]



Sunday October, 17th.

Theme 4: Synaptic Transmission and Cell Signalling


Pyotr Bregestovski
Institut de Neurobiologie de la Mediterranee (INMED), Marseille, France


Glycinergic synaptic transmission: modulation by calcium and endocannabinoids

The entry of Ca2+ into neurons provides a major signal for the regulation of Ca2+-dependent proteins and contributes to a number physiological and pathological processes, including synapse formation, the induction of various forms of synaptic plasticity and cell death. Over the past years, there has been remarkable progress in defining novel implications of Ca2+ and Ca2+-biding proteins in modulation of synaptic transmission. Using glycinergic synapse as the main example, two main areas of novel observations will be discussed: (i) modulation of postsynaptic ionic channels by Ca2+-binding proteins and (ii) modulation of neurotransmitter release from presynaptic terminals by Ca2+-dependent retrograde messengers, endocannabinoids (see rev. Wilson & Nicols, 2002; Diana, Marty, 2004). Other mechanisms of Ca-induced short-term plasticity will be also discussed.
Glycine receptors, mediating inhibition in spinal cord and brainstem, are modulated by intracellular Ca2+, whose increase leads to the fast potentiation of glycine-activated currents (Fucile et al., 2000). In heterologous systems this potentiation develops in less than 100 ms and it is characterized by an increase of sensitivity to glycine. To understand the physiological relevance of this Ca2+-dependent modulation, we analyzed the effects of intracellular Ca2+ increase on glycinergic synaptic currents in visually identified motoneurons of the rat brainstem hypoglossal nucleus.
Our findings demonstrate two independent mechanisms of Ca2+-induced modulation of glycinergic synaptic transmission in brainstem motoneurons: (i) the cannabinoid-dependent presynaptic suppression of neurotransmitter release; (ii) potentiation of postsynaptic GlyR currents. While presynaptic suppression is a prevailing phenomenon in HMs, Ca2+-dependent potentiation represents a novel mechanism of control of glycinergic transmission, whose relevance needs to be elucidated. Rerefences.

  1. Diana MA, Marty A. (2004) Endocannabinoid-mediated short-term synaptic plasticity: depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE). Br J Pharmacol.
  2. Fucile, S., D. De Saint Jan, et al. (2000). Fast potentiation of glycine receptor channels of intracellular calcium in neurons and transfected cells. Neuron 28(2): 571-83. [PDF]
  3. Wilson, R. I. and R. A. Nicoll (2002). Endocannabinoid signaling in the brain. Science 296(5568): 678-82. [PDF]


V.I. Govardovsky
Institute for Evolutionary Physiology & Biochemistry, Russian Academy of Sciences 194223 St. Petersburg, Russia


Regulation of the phototransduction cascade

Phototransduction is the process by which the absorption of light by a visual pigment molecule is converted into electrical response of a photoreceptive cell. Visual pigment of retinal photoreceptor cells, rhodopsin, belongs to the family of seven-helical transmembrane receptors that transmit signal via G-proteins. Notably, the system is used in several other sensory systems, for example in olfaction, but also serves for reception of some hormones and neurotransmitters engaged in regulation of muscle function, blood flow and synaptic transmission. Due to a number of practical reasons, phototransduction is the best studied among them, so the data obtained on retinal rods and cones provide a wealth of information about other similar systems [1].
A generalized G-protein-signaling chain consists of a receptor molecule, a G-protein, an effector enzyme, and an intracellular messenger. In vertebrate photoreceptor cells, the messenger is cGMP, and the effector enzyme, cGMP phosphodiesterase (PDE). Absorption of light by rhodopsin leads via photoreceptor-specific G-protein transducin (T) to activation of PDE and to decrease in cGMP concentration that directly controls the conductance of ionic channels of the plasma membrane thus producing an electrical signal. The phototransduction cascade provides two-step enzymatic amplification. Firstly, light-activated rhodopsin catalyses the exchange of GDP for GTP on few hundreds of Ts. Secondly, active T-GTP-PDE complexes hydrolyze each several hundreds of cGMPs. High gain of the cascade allows retinal rods to reliably signal absorptions of single photons. However, high sensitivity comes at price; it makes photoreceptors susceptible to saturation in daylight. Thus retinal photoreceptors exploit an efficient system of regulation, called light adaptation, which adjusts their speed and sensitivity accordingly to ambient light level [2].
Three best-studied mechanisms of light adaptation operate via light-induced changes of cytoplasmic Ca2+ concentration. Closure of cGMP-gated channels in light blocks calcium influx thus lowering [Ca2+]in. Decrease in [Ca2+]in shortens the life-time of activated rhodopsin by speeding up its phosphorylation, activates guanyl cyclase to oppose accelerated hydrolysis of cGMP, and increases the affinity of cGMP-gated channels to cGMP. Concerted operation of the three loops of the negative feedback expands the range of working light intensities by 2 to 3 orders of magnitude. All molecular components of the feedback system are rather well characterized and function of each is proven in corresponding knockout animals and direct biochemical and physiological experiments. Thus vertebrate transduction and its regulation are studied in unprecedented details [1].
However, recent data show that some important mechanisms are still missing from the existing scheme. It seems that there are other, yet unknown ways of quickly modifying properties of the components of the cascade to further extend its operating range. Besides, there are regulatory mechanisms that rely upon massive transport of proteins between cellular compartments within photoreceptors. They result in long-lasting changes of cascade amplification and kinetics [3]. The emerging picture is far more complex than appeared just five years ago, and there is no doubt that we can await further surprises from the apparently perfectly studied system.
[1] Pugh E.N., Jr. & Lamb T.D. (2000). Phototransduction in vertebrate rods and cones: Molecular mechanisms of amplification, recovery and light adaptation. In: D.G. Stavenga, W.J. de Grip, & E.N.Pugh, Jr. (Eds.), Handbook of Biological Physics, 3, Chapter 5 (pp. 183-255). Amsterdam: Elsevier Science B.V.
[2] Govardovskii V.I., Calvert P.D., Arshavsky V.Y. Photoreceptor light adaptation: Untangling desensitization and sensitization // J. Gen. Physiol. 2000. V. 116. p. 791-794. [PDF]
[3] M.Sokolov, A.Lyubarsky, K.J.Strissel, A.Savchenko, V.I. Govardovskii, E.N. Pugh, Jr. and V.Y. Arshavsky. Massive light-dependent translocation of transducin between the functional compartments of rod photoreceptors: a novel mechanism of light adaptation // Neuron. 2002. V. 33. p. 95 - 106. [PDF]


Coffee break


Elena Kaznacheyeva, Gusev K., Nikolaev A., Mozhayeva G.N.
Institute of Cytology RAS, St-Petersburg, Russia


Store operated calcium channels in non-excitable cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of Ca2+ from intracellular Ca2+ stores and promotes Ca2+ influx across the plasma membrane via capacitative Ca2+ entry (CCE) or store-operated Ca2+ entry (SOC) processes [1]. Two types of Ca2+ currents have been implicated in store-operated Ca2+ entry in non-excitable cells. Highly Ca2+-selective and inwardly rectifying current through very low conductance channels (24 fS) named "Ca2+ release activated calcium current" (ICRAC) have been initially discovered in studies of Jurkat and RBL cells. Ca2+ currents with less selectivity and higher single channel conductance have been later identified in a number of cells and grouped under the name ISOC. The molecular identity of ICRAC remains unclear. Channels of mammalian TRPC family are the most likely candidates for the role of ISOC channels. The mechanisms of ISOC and ICRAC channel activation have been under intense investigation. Activation of ISOC/ICRAC channels by a diffusible messenger CIF (Ca2+-influx factor) released by depleted Ca2+ stores, via "conformational coupling" with the intracellular InsP3R, and by regulated insertion of channels into plasma membrane have been considered. However, the nature of store-derived signal to activate SOCs remains unresolved [2].
In experiments with a human carcinoma A431 cell line we described low conductance plasma membrane Ca2+ channels (Imin) activated by stimulation of PLC-coupled surface receptors or passive store depletion. Imin activity is induced in excised inside-out patches by addition of IP3. N-terminal of IP3R has been shown to be responsible for gating of Imin. We found that activation of Imin channels by InsP3 in inside-out patches is facilitated by anti-PIP2 antibodies. These findings led us to suggestion of existence of InsP3R-PIP2-Imin signaling complex in these cells [3]. Using whole-cell recordings we have found two types of store-operated currents in A431 cells - highly selective ICRAC and less selective ISOC supported by Imin [4].
Supported by SS-2178.2003.4, RFBR 04-04-49053, 04-04-49057, the program of "Molecular and cell biology". References

  1. A.B Parekh and R. Penner (1997), Physiol. Rev. 77, 901-903
  2. K. Venkatachalam et al., (2002), Nat.Cell.Biol. 4, 263-272 [PDF]
  3. E. Kaznacheyeva et al., (2001), Proc.Natl.Acad.Sci. USA 98, 148-153 [PDF]
  4. K. Gusev et al., (2003), J.Gen.Physiol. 122, 81-94 [PDF]


Vsevolod Tkachuk Alexander Vorotnikov
Cardiology Research Center, Moscow 121552, Russia


Ca-dependent and independent pathways in cell motility and contractility

Ca2+ is a key intracellular messenger to activate actomyosin, smooth muscle cell contractility and motility. In association with calmodulin it activates myosin light chain kinase (MLCK), which switches on myosin by phosphorylating Ser19 of its regulatory light chain (RLC), and removes inhibition of actin filaments imposed by caldesmon. However, the long standing paradigm that only Ca2+ is required for actomyosin activation became outdated since a discovery of several Ca2+-independent pathways that target both myosin RLC and caldesmon. Here the data will be presented on the role of protein kinase C and Rho-kinase in stimulation of vascular smooth muscle cell contraction and motility. We show that smooth muscle tonic contraction can be induced in the absence of Ca2+ by selective consequent activation of protein kinase C and MAP-kinases that target caldesmon and MLCK. Similarly, the vascular smooth muscle cell chemotaxis is stimulated in the absence of Ca2+ by urokinase type plasminogen activator, which activates MAP-kinases and Rho-kinase signalling pathways to target myosin RLC and caldesmon phosphorylation. We identified critical sites phosphorylated in MLCK and caldesmon and revealed biochemical effects of their phosphorylation. We suggest that MAP-kinase pathway is involved in Ca2+-independent actomyosin activation and may cooperate with the Ca2+-dependent mechanisms at the level of actomyosin-associated regulatory proteins.

  1. Goncharova et al. (in press) Activation of p38 MAP-kinase and caldesmon phosphorylation are essential for urokinase-induced human smooth muscle cell migration [PDF]
  2. Mukhina et al. (2000) J. Biol. Chem., 275 (22)16450-16458. The Chemotactic Action of Urokinase on Smooth Muscle Cells Is Dependent on Its Kringle Domain [PDF]


Concluding remarks