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PubMed2005

The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity.

The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively. We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (I24C-SL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin-spin interaction with the neighboring I24C-SL residues in the MscL channel pentamer. The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity.

Arginine↗

PubMed2005

Coupling actin dynamics to the endocytic process in Saccharomyces cerevisiae.

Endocytosis is an essential eukaryotic process that, in many systems, has been reported to require a functional actin cytoskeleton. The process of endocytosis is critical for controlling the protein-lipid composition of the plasma membrane and uptake of nutrients as well as pathogens and also plays an important role in regulation of cell signalling. While several distinct pathways for endocytosis have been characterised, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly supported by studies in mammalian cells. Current evidence suggests that cortical patches are sites of endocytosis in Saccharomyces cerevisiae and that these sites are composed of sequentially forming protein complexes. Distinct stages in complex formation are characterised by the presence of different activators of F-actin polymerisation. Disassembly of the complexes is also essential for the endocytosis to proceed. Mutants lacking the kinases Ark1 and Prk1 accumulate actin and endocytic machinery in a single large clump in cells. Phosphorylation of endocytic proteins including Sla1p is proposed to cause their removal from the complex and allow later stages of the invagination process to occur. Dephosphorylation of endocytic components may then allow subsequent reincorporation into new sites of endocytic complex assembly.

Actins↗

PubMed1987

Single low-voltage-activated calcium channels in chick and rat sensory neurones.

1. Single and multiple Ca2+ channel currents were recorded from outside-out and cell-attached patches of cultured chick and rat dorsal root ganglion cells, using the patch-clamp technique. 2. Outside-out patches containing a large number of Ca2+ channels revealed Ca2+ currents resembling those from the whole cell. A low-voltage-activated (l.v.a.) and a high-voltage-activated (h.v.a.) Ca2+ current similar to those described in the accompanying paper (Carbone & Lux, 1987 b) could be distinguished. The h.v.a. current component subsided within 10 min following the formation of the patch, while the l.v.a. component lasted much longer. 3. Unitary events related to the l.v.a. Ca2+ channel could be clearly resolved in outside-out patches formed in Na+- and K+-free media containing 5-50 mM-CaCl2. 4. The amplitudes of l.v.a. channel openings were bimodally distributed, indicating the presence of two conductive states. At -40 mV, mean amplitudes of the two events were -0.29 +/- 0.07 pA and -0.47 +/- 0.085 pA in 50 mM-CaCl2, with apparent slope conductances of about 3.6 and 5.2 pS, respectively. In 5 mM-CaCl2 both slope conductances were about 3 times smaller. The mean open times were similar for both states and were fitted by a simple exponential with a time constant of about 2.5 ms at -40 mV. The time constant decreased with more-negative membrane potentials and was 0.9 ms at -100 mV. Openings frequently occurred in bursts separated by longer-lasting closures. The mean closed time during bursts was 1.33 ms at -40 mV. 5. Time and amplitude distributions of elementary events were similar for chick and rat sensory neurones and with Ba2+ and Sr2+ replacing external Ca2+. 6. In the potential range examined (from -60 to -30 mV), the first-latency distribution function revealed a distinct rise to peak which occurred at considerably earlier times than peaks of macroscopic currents. The time course of macroscopic l.v.a. Ca2+ currents could be simulated in two ways: (a) by using a five-state Markov-chain model with rate constants estimated from the transition probabilities and dwell times of the channel states, and (b) by evaluating the convolution integral of the first-latency function and the burst open probability of the channel. Both approaches suggest that activation and inactivation are weakly coupled and that the l.v.a. channel of sensory neurones reopens several times before inactivating.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed2006

StdpC: a modern dynamic clamp.

With the advancement of computer technology many novel uses of dynamic clamp have become possible. We have added new features to our dynamic clamp software StdpC ("Spike timing-dependent plasticity Clamp") allowing such new applications while conserving the ease of use and installation of the popular earlier Dynclamp 2/4 package. Here, we introduce the new features of a waveform generator, freely programmable Hodgkin-Huxley conductances, learning synapses, graphic data displays, and a powerful scripting mechanism and discuss examples of experiments using these features. In the first example we built and 'voltage clamped' a conductance based model cell from a passive resistor-capacitor (RC) circuit using the dynamic clamp software to generate the voltage-dependent currents. In the second example we coupled our new spike generator through a burst detection/burst generation mechanism in a phase-dependent way to a neuron in a central pattern generator and dissected the subtle interaction between neurons, which seems to implement an information transfer through intraburst spike patterns. In the third example, making use of the new plasticity mechanism for simulated synapses, we analyzed the effect of spike timing-dependent plasticity (STDP) on synchronization revealing considerable enhancement of the entrainment of a post-synaptic neuron by a periodic spike train. These examples illustrate that with modern dynamic clamp software like StdpC, the dynamic clamp has developed beyond the mere introduction of artificial synapses or ionic conductances into neurons to a universal research tool, which might well become a standard instrument of modern electrophysiology.

Action Potentials↗

PubMed1992

Gap junctional channels in adult mammalian sinus nodal cells. Immunolocalization and electrophysiology.

The subcellular mechanism of cell-to-cell communication in the natural pacemaker region of the mammalian heart was studied using electrophysiological and immunofluorescence techniques in isolated pairs of rabbit sinus nodal cells. By measuring whole-cell currents using a double patch-clamp approach, it was demonstrated that communication in the sinus node is mediated through gap junctional channels similar to those in other types of adult cardiac cell pairs. Macroscopic sinus nodal junctional resistance had a mean value of 387.9 +/- 97.1 M omega (mean +/- SEM, n = 10) and was greatly increased by superfusion with alkanols. Single-channel junctional conductance could be resolved in three cell pairs. Given their high membrane resistance (1.16 +/- 0.32 G omega, n = 12), the electrical coupling provided by as few as three gap junctional channels between nodal cells will allow for pacemaker synchronization. Further evidence for the presence of the channels was obtained from immunofluorescent double-labeling of desmin and the gap junction protein (connexin43) in sinus nodal tissue as well as in cultured sinus nodal cells. Using antisera against residues 243-257 of the connexin43 protein, a specific staining at the site of cell-to-cell apposition was demonstrated. These data provide direct evidence in favor of electronic coupling as the means for achieving pacemaker synchronization in the rabbit sinus node.

Animals↗

PubMed2005

Bistable network behavior of layer I interneurons in auditory cortex.

GABAergic interneurons in many areas of the neocortex are mutually connected via chemical and electrical synapses. Previous computational studies have explored how these coupling parameters influence the firing patterns of interneuronal networks. These models have predicted that the stable states of such interneuronal networks will be either synchrony (near zero phase lag) or antisynchrony (phase lag near one-half of the interspike interval), depending on network connectivity and firing rates. In certain parameter regimens, the network can be bistable, settling into either stable state depending on the initial conditions. Here, we investigated how connectivity parameters influence spike patterns in paired recordings from layer I interneurons in brain slices from juvenile mice. Observed properties of chemical and electrical synapses were used to simulate connections between uncoupled cells via dynamic clamp. In uncoupled pairs, action potentials induced by constant depolarizing currents had randomly distributed phase differences between the two cells. When coupled with simulated chemical (inhibitory) synapses, however, these pairs exhibited a bimodal firing pattern, tending to fire either in synchrony or in antisynchrony. Combining electrical with chemical synapses, prolonging tau(Decay) of inhibitory connections, or increasing the firing rate of the network all resulted in enhanced stability of the synchronous state. Thus, electrical and inhibitory synaptic coupling constrain the relative timing of spikes in a two-cell network to, at most, two stable states, the stability and precision of which depend on the exact parameters of coupling.

Animals↗

PubMed1994

Three-dimensional structure in solution of neurotoxin III from the sea anemone Anemonia sulcata.

The three-dimensional structure in aqueous solution of the 27-residue polypeptide neurotoxin Anemonia sulcata toxin III (ATX III) has been determined from 1H NMR data. As ATX III self-associates in the millimolar concentration range, causing a marked concentration dependence for the chemical shifts of several residues [Norton, R. S., Cross, K., Braach-Maksvytis, V., & Wachter, E. (1993) Biochem. J. 293, 545-551], it was necessary to record NOESY spectra over a range of concentrations in order to eliminate any intermolecular interactions from the NOE restraint set. The pairings of the six half-cystine residues were also unknown and had to be determined (as 3-17, 4-11, and 6-22) from preliminary structure calculations performed using both upper bound distance restraints from NOESY data and a substantial number of lower bound restraints inferred from the absence of NOESY cross-peaks. Final structures were determined, using the program X-PLOR, from interproton distance restraints inferred from NOEs, backbone and side chain dihedral angle restraints from spin-spin coupling measurements, and a smaller number of lower bound restraints. Stereospecific assignments for 11 beta-methylene pairs were also included. The final set of 28 structures had an average pairwise RMS difference of 1.32 A over the backbone heavy atoms (N, C alpha, and C) and 2.18 A over all heavy atoms. For the well-defined region encompassing residues 3-22, the corresponding values were 0.62 and 1.28 A, respectively. ATX III adopts a compact structure containing four reverse turns (a distorted type I beta-turn at residues 6-9, a type I beta-turn at residues 8-11, and inverse gamma-turns at residues 12-14 and 15-17) and two other chain reversals, but no regular alpha-helix or beta-sheet. Several of the residues most affected by aggregation are located on the surface of the molecule, forming a hydrophobic patch which may constitute part of the sodium channel binding surface. Possible relationships between the structure of ATX III and those of other sea anemone toxins that interact with the same site on the voltage-gated sodium channel are considered.

Amino Acid Sequence↗

PubMed1998

Overexpression of nerve growth factor in the heart alters ion channel activity and beta-adrenergic signalling in an adult transgenic mouse.

1. The electrophysiological and pharmacological properties of cardiac myocytes from the hearts of adult transgenic mice engineered to overexpress nerve growth factor (NGF) in the heart were studied. 2. There was a 12% increase in the ventricular myocyte capacitance in NGF myocytes consistent with cardiac hypertrophy, and action potential duration at 90% repolarization (APD90) was prolonged by 142 % compared with wild-type (WT) myocytes. This was due, at least in part, to a decrease in the density of two K+ currents, Ito and IK(ur), which were significantly reduced in NGF mice with no change in their electrophysiological characteristics. We found no change in the current density or electrophysiological properties of the L-type Ca2+ current. 3. The effect on Ito and IK(ur) of TEA and 4-aminopyridine (4-AP) was not different in cells isolated from WT and NGF mice. The prolongation of APD observed in NGF cells was mimicked in WT cells by exposure to 1 mM 4-AP, which partially blocked Ito, completely blocked IK(ur) and increased APD90 by 157%. 4. The isoprenaline-induced increase in ICa was significantly smaller in NGF myocytes than in WT myocytes. This was not due to a decrease in beta-adrenergic receptor (beta-AR) density, as this was increased in NGF tissue by 55%. Analysis of beta-AR subtypes showed that this increase was entirely due to an increase in beta2-AR density with no change in beta1-ARs. 5. The response of the beta-AR-coupled adenylyl cyclase system to isoprenaline, Gpp(NH)p and forskolin was studied by measuring cAMP production. In NGF tissue, isoprenaline elicited a significantly smaller response than in WT myoyctes and this was not due to reduced adenylyl cyclase activity as the responses of NGF tissue to guanylylimidodiphosphate (Gpp(NH)p) and forskolin were unaffected. 6. In conclusion, the overexpression of NGF in the mouse heart resulted in a decrease in the current density of two K+ channels, which contributed to the prolongation of the cardiac action potential. Despite an increase in beta2-AR density in the hearts of the NGF mice, the response to isoprenaline was diminished, and this was due to an uncoupling of the beta-ARs from the intracellular signalling cascade. These potentially pathological changes may be involved in the occurrence of ventricular arrhythmias in cardiac hypertrophy and failure, and this mouse provides a novel model in which to study such changes.

Action Potentials↗

PubMed1998

Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity.

The designed G120R mutant of human growth hormone (hGH) is an antagonist and can bind only one molecule of the growth hormone receptor. We have determined the crystal structure of the 1:1 complex between this mutant and the receptor extracellular domain (hGHbp) at 2.6 A resolution, and used it to guide a detailed survey of the structural and functional basis for hormone-receptor recognition. The overall structure of the complex is very similar to the equivalent portion of the 1:2 complex, showing that formation of the active complex does not involve major conformational changes. However, a segment involved in receptor-receptor interactions in the 1:2 complex is disordered in this structure, suggesting that its productive conformation is stabilized by receptor dimerization. The hormone binding site of the receptor comprises a central hydrophobic patch dominated by Trp104 and Trp169, surrounded by a hydrophilic periphery containing several well-ordered water molecules. Previous alanine scanning showed that the hydrophobic "hot spot" confers most of the binding energy. The new structural data, coupled with binding and kinetic analysis of further mutants, indicate that the hot spot is assembled cooperatively and that many residues contribute indirectly to binding. Several hydrophobic residues serve to orient the key tryptophan residues; kinetic analysis suggests that Pro106 locks the Trp104 main-chain into a required conformation. The electrostatic contacts of Arg43 to hGH are less important than the intramolecular packing of its alkyl chain with Trp169. The true functional epitope that directly contributes binding energy may therefore comprise as few as six side-chains, participating mostly in alkyl-aromatic stacking interactions. Outside the functional epitope, multiple mutation of residues to alanine resulted in non-additive increases in affinity: up to tenfold for a hepta-alanine mutant. Contacts in the epitope periphery can therefore attenuate the affinity of the central hot spot, perhaps reflecting a role in conferring specificity to the interaction.

Binding Sites↗

PubMed2005

M3-like muscarinic receptors mediate Ca2+ influx in rat mesencephalic GABAergic neurones through a protein kinase C-dependent mechanism.

GABAergic neurones in the mesencephalon are important regulators of dopamine neurones. Cholinergic projections from mesopontine nuclei preferentially synapse onto these GABAergic neurones, thus suggesting that ACh can regulate dopamine neurones indirectly by modulating GABAergic interneurones. Muscarinic receptors mediate excitation of these interneurones through a Ca(2+)-dependent mechanism. Using a mesencephalic primary culture model, we show here that muscarine (10 microM) increases intracellular Ca2+ concentrations ([Ca2+]i) in GABAergic interneurones. Compatible with previous anatomical data, our pharmacological studies further suggest that the M3 receptor is the primary mediator of this increase. The rise in [Ca2+]i induced by muscarine was not activity-dependent but required influx of Ca2+ from the extracellular medium. Consistent with the known coupling of the M3 receptor to PKC, the effect of muscarine was blocked by bisindolylmaleimide, a selective PKC antagonist. The effect of muscarine was inhibited by SKF 96365 and verapamil, drugs known to block non-selective cationic channels such as those formed by transient receptor potential (TRPC) proteins. Finally, GABAergic neurones were found to be immunopositive for TRPC1, 3, 5 and 6. Taken together, these results suggest that the Ca(2+)-dependent regulation of mesencephalic GABAergic neurones by muscarinic receptors requires activation of some receptor-operated Ca2+ channels through a PKC-dependent mechanism.

Analysis of Variance↗

PubMedDate not supplied

Cyclic GMP can increase rod outer-segment light-sensitive current 10-fold without delay of excitation.

To test the hypothesis that cyclic GMP is the internal messenger coupling rhodopsin activation to membrane excitation in vertebrate rod photoreceptors, we used a novel technique combining measurement of membrane currents of isolated salamander rods with a suction electrode and the introduction of cyclic GMP through a whole-cell recording patch pipette. Rupture of an attached patch was followed by a rapid (approximately 10 s), approximately 10-fold increase in outer-segment membrane current, all of which was light-sensitive. There was little change in the rising phase of the response to a saturating flash, but the duration of the saturated phase of the response increased approximately 10-fold. The effects reversed completely within 3-4 min after withdrawal of the cyclic GMP-containing patch pipette. A formal kinetic analysis shows that the first two observations are inconsistent with the postulate that cyclic GMP opens the light-sensitive conductance by simple binding to channels, unless free cyclic GMP in the outer segment is assumed to be much lower than published estimates, and most of the outer-segment cyclic GMP is bound and inexchangeable on the timescale of 200 ms. Furthermore, our results suggest that rod cyclic GMP is not involved solely in keeping the light-sensitive conductance open, but may also affect the activity of the phosphodiesterase that mediates cyclic GMP hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2004

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids.

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a beta-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.

Amino Acid Sequence↗

PubMed2003

Maturation of EPSCs and intrinsic membrane properties enhances precision at a cerebellar synapse.

The timing of action potentials is an important determinant of information coding in the brain. The shape of the EPSP has a key influence on the temporal precision of spike generation. Here we use dynamic clamp recording and passive neuronal models to study how developmental changes in synaptic conductance waveform and intrinsic membrane properties combine to affect the EPSP and action potential generation in cerebellar granule cells. We recorded EPSCs at newly formed and mature mossy fiber-granule cell synapses. Both quantal and evoked currents showed a marked speeding of the AMPA receptor-mediated component. We also found evidence for age- and activity-dependent changes in the involvement of NMDA receptors. Although AMPA and NMDA receptors contributed to quantal EPSCs at immature synapses, multiquantal release was required to activate NMDA receptors at mature synapses, suggesting a developmental redistribution of NMDA receptors. These changes in the synaptic conductance waveform result in a faster rising EPSP and reduced spike latency in mature granule cells. Mature granule cells also have a significantly decreased input resistance, contributing to a faster decaying EPSP and a reduced spike jitter. We suggest that these concurrent developmental changes, which increase the temporal precision of EPSP-spike coupling, will increase the fidelity with which sensory information is processed within the input layer of the cerebellar cortex.

Action Potentials↗

PubMed2000

Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA.

Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.

Amino Acid Motifs↗

PubMed2006

The novel antidyskinetic drug sarizotan elicits different functional responses at human D2-like dopamine receptors.

Sarizotan (EMD 128130) is a chromane derivative that exhibits affinity at serotonin and dopamine receptors. Sarizotan effectively suppresses levodopa-induced dyskinesia in primate and rodent models of Parkinson's disease, and tardive dyskinesia in a rodent model. Results from clinical trials suggest that sarizotan significantly alleviates levodopa-induced dyskinesia. The functional effects of sarizotan on individual dopamine receptor subtypes are not known. Here we report the functional effects of sarizotan on human D2-like dopamine receptors (D2S, D2L, D3, D4.2 and D4.4) individually expressed in the AtT-20 neuroendocrine cell line. Using the coupling of D2-like dopamine receptors to G-protein coupled inward rectifier potassium channels we determined that sarizotan is a full agonist at D3 and D4.4 receptors (EC50=5.6 and 5.4 nM, respectively) but a partial agonist at D2S, D2L and D4.2 receptors (EC50=29, 23 and 4.5 nM, respectively). Consistent with its partial agonist property, sarizotan is an antagonist at D2S and D2L receptors (IC50=52 and 121 nM, respectively). Using the coupling of D2-like dopamine receptors to adenylyl cyclase we determined that sarizotan is a full agonist at D2L, D3, D4.2 and D4.4 receptors (EC50=0.51, 0.47, 0.48 and 0.23 nM, respectively) but a partial agonist at D2S receptors (EC50=0.6 nM).

Animals↗

PubMed1993

Voltage-dependent calcium channel current in isolated gallbladder smooth muscle cells of guinea pig.

The voltage-dependent Ca2+ current was studied in enzymatically dispersed guinea pig gallbladder smooth muscle cells using the whole cell patch-clamp technique. Depolarizing voltage (V) steps induced an inward current (I) that was carried by Ca2+. The threshold potential was -40 to -30 mV, the maximal current was observed at +10 to +20 mV, and the reversal potential was around +80 mV. I-V curves obtained with holding potentials of -80 and -40 mV were not significantly different. This current had a high sensitivity to dihydropyridine drugs, and the Ba2+ or Sr2+ current was larger than the Ca2+ current. Activation was accelerated by increasing the membrane potential. In general, the time course of decay was well fitted by the sum of two exponentials, but consideration of a third (ultra-slow) decay component was also necessary when the current generated by a 2-s command pulse was analyzed. Superimposition of activation and inactivation curves showed the presence of a significant window current. Carbachol suppressed the Ca2+ current only when the pipette contained a low concentration of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. These results show that the L-type Ca2+ current is dominant in gallbladder smooth muscle cells and may contribute to excitation-contraction coupling.

Animals↗

PubMed2006

Inherited cortical HCN1 channel loss amplifies dendritic calcium electrogenesis and burst firing in a rat absence epilepsy model.

While idiopathic generalized epilepsies are thought to evolve from temporal highly synchronized oscillations between thalamic and cortical networks, their cellular basis remains poorly understood. Here we show in a genetic rat model of absence epilepsy (WAG/Rij) that a rapid decline in expression of hyperpolarization-activated cyclic-nucleotide gated (HCN1) channels (I(h)) precedes the onset of seizures, suggesting that the loss of HCN1 channel expression is inherited rather than acquired. Loss of HCN1 occurs primarily in the apical dendrites of layer 5 pyramidal neurons in the cortex, leading to a spatially uniform 2-fold reduction in dendritic HCN current throughout the entire somato-dendritic axis. Dual whole-cell recordings from the soma and apical dendrites demonstrate that loss of HCN1 increases somato-dendritic coupling and significantly reduces the frequency threshold for generation of dendritic Ca2+ spikes by backpropagating action potentials. As a result of increased dendritic Ca2+ electrogenesis a large population of WAG/Rij layer 5 neurons showed intrinsic high-frequency burst firing. Using morphologically realistic models of layer 5 pyramidal neurons from control Wistar and WAG/Rij animals we show that the experimentally observed loss of dendritic I(h) recruits dendritic Ca2+ channels to amplify action potential-triggered dendritic Ca2+ spikes and increase burst firing. Thus, loss of function of dendritic HCN1 channels in layer 5 pyramidal neurons provides a somato-dendritic mechanism for increasing the synchronization of cortical output, and is therefore likely to play an important role in the generation of absence seizures.

Action Potentials↗

PubMed1999

ATP sensitive potassium channel and myocardial preconditioning.

KATP channels play an important role in physiology and pathophysiology of many tissues. As in the pancreatic beta cells, they couple the change of blood glucose with insulin release. The data coming from Baukrowitz et al. and Shyng and Nichols gave the possible answers to the two old enigmas of KATP channels, i.e., different ATP sensitivity reported in the same tissue and how the channel opened under intracellular millimolar ATP concentration, in which they showed the lipids and lipid metabolites are essential for KATP channel regulation by altering ATP sensitivity. This new information rises several further considerations. How does PIP2 reduce the sensitivity of the channel to ATP? In order to clarify the possibility of direct competing or allosteric effect on the ATP binding site, competitive binding assay should be performed. Since the PIP2 theory seems to be the key event to determine the ATP sensitivity and thus control the channel open probability, then what is the resting concentration of PIP2 in the cell membrane? Is it sufficient to account for the difference in the ATP sensitivity of the intact cell and excised patch from different tissues? Quantitative studies either immunoblotting by PIP2 antibody or fluorescence-labeled lipid assay-may obtain some basic but useful data for further studies to answer these questions. Furthermore, the ATPi mediated restoration of activity was inhibited by antibodies against PIP2. The dualistic behavior of KATP channels to intracellular NDPs should be reexamined with respect to PIP2. The vast majority of preconditioning studies has been performed in intact animals in which myocardial infarct size was used as the end point to define the cardio-protective effect of ischemic PC. These results suggest a key role for the KATP channel as both a trigger and as an end effector of both acute and delayed ischemic PC. The persistent activation of KATP channels during the early reperfusion phase is essential for a smooth and full recovery of contractile function, as well as for maintenance of electrical stability in heart that has been exposed to ischemia. Though activate adenosine A1 receptor coupled with Gi protein can open the KATP channels, adenosine is quickly released during ischemia and exerts potent coronary vasodilatation to maintain coronary blood flow through A2 receptors. This adenosine-induced coronary vasodilatation could be coupled with KATP channels based on the evidence of the augmentation effect of KCOs. Nitric oxide may also play some role in both first and second window of myocardial protection. It is possible that rapid and reversible phosphorylation and activation of constitutive expressed myocardial NOS or by direct KATP channel phosphorylation and activation leads to the first window of myocardial protection. This hypothesis can be further investigated either by using site direct mutagenesis of iNOS or KATP channel, or by applying the dominant negative iNOS in the cell ischemic model, or by building the adenosine or iNOS knock-out mice to study the relationship of these possible mechanisms. Recently, Kontos further showed that KCOs need L-lysine or L-arginine to dilate cerebral arterioles. This suggests that there may be an amino acid binding site inside the KATP channel and nitric oxide can open the KATP channel either by direct acting on the channel protein or by modulating the affinity of the amino acid binding site for L-lysine or L-arginine. Other KATP channel openers in need of additional characterization are the Type III KCOs (nicorandiol). They open the KATP channel only in the presence of elevated intracellular NDPs, which may make them specifically target to the ischemic region, because the intracellular NDP increases mostly in ischemic region. It is possible that type III KCOs can selectively improve blood flow to ischemic areas without diverting blood away to non-ischemic region, and prevents the "steal phenomenon". (ABSTRACT TRUNCATED)

Adenosine Triphosphate↗