A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were... more A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, G~ was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary unitary channel conductance junctional currents was detected corresponding to an " of ~ 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic G~ voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.
Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinc... more Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinct diseases associated with sudden death: one form of the long-QT syndrome (LQT 3 ) and the Brugada syndrome. We have screened SCN5A in a large 8-generation kindred characterized by a high incidence of nocturnal sudden death, and QT-interval prolongation and the "Brugada ECG" occurring in the same subjects. An insertion of 3 nucleotides (TGA) at position 5537, predicted to cause an insertion of aspartic acid (1795insD) in the C-terminal domain of the protein, was linked to the phenotype and was identified in all electrocardiographically affected family members. ECGs were obtained from 79 adults with a defined genetic status (carriers, nϭ43; noncarriers, nϭ36). In affected individuals, PR and QRS durations and QT intervals are prolonged (PϽ0.0001 for all parameters). ST segment elevation in the right precordial leads is present as well (PϽ0.0001). Twenty-five family members died suddenly, 16 of them during the night. Expression of wild-type and mutant Na ϩ channels in Xenopus oocytes revealed that the 1795insD mutation gives rise to a 7.3-mV negative shift of the steady-state inactivation curve and an 8.1-mV positive shift of the steady-state activation curve. The functional consequence of both shifts is likely to be a reduced Na ϩ current during the upstroke of the action potential. LQT 3 and Brugada syndrome are allelic disorders but may also share a common genotype. (Circ Res. 1999;85:1206-1213.)
American Journal of Physiology Heart and Circulatory Physiology, May 1, 2001
In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchron... more In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.
Biochemical and Biophysical Research Communications, Oct 20, 2006
Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven... more Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays >60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.
ABSTRACT The main feature of gap junctions is the passage of ions and small molecules between adj... more ABSTRACT The main feature of gap junctions is the passage of ions and small molecules between adjacent cells. In the heart, for example, junctional currents between adjacent cells are elicited by the membrane potential difference when one of the cells generates an action potential, while the other cell is still at resting membrane potential, thereby allowing propagation of the action potential. Experimentally, such gap junctional currents can be directly evoked and measured by independently controlling the membrane voltage of both cells of a cell pair. This is accomplished by “dual voltage clamping” a cell-pair, a method introduced by Spray et al. (1). Using separate electrodes for voltage control and current injection, this technique allowed accurate determination of kinetics and conductance of gap junctions (1–3). At present, the dual voltage-clamp technique is usually performed with two patch pipets (4), each on one cell of a cell pair, which control voltage and measure current simultaneously (5–8).
Journal of Molecular and Cellular Cardiology, 2003
We previously described a Dutch family in which congenital cardiac conduction disorder has clinic... more We previously described a Dutch family in which congenital cardiac conduction disorder has clinically been identified. The ECG of the index patient showed a first-degree AV block associated with extensive ventricular conduction delay. Sequencing of the SCN5A locus coding for the human cardiac Na+ channel revealed a single nucleotide deletion at position 5280, resulting in a frame-shift in the sequence coding for the pore region of domain IV and a premature stop codon at the C-terminus. Wild type and mutant Na+ channel proteins were expressed in Xenopus laevis oocytes and in mammalian cells. Voltage clamp experiments demonstrated the presence of fast activating and inactivating inward currents in cells expressing the wild type channel alone or in combination with the beta1 subinut (SCN1B). In contrast, cells expressing the mutant channels did not show any activation of inward current with or without the beta1 subunit. Culturing transfected cells at 25 degrees C did not restore the Na+ channel activity of the mutant protein. Transient expression of WT and mutant Na+ channels in the form of GFP fusion proteins in COS-7 cells indicated protein expression in the cytosol. But in contrast to WT channels were not associated with the plasma membrane. The SCN5A/5280delG mutation results in the translation into non-function channel proteins that do not reach the plasma membrane. This could explain the cardiac conduction defects in patients carrying the mutation.
A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were... more A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, G~ was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary unitary channel conductance junctional currents was detected corresponding to an " of ~ 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic G~ voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.
Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinc... more Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinct diseases associated with sudden death: one form of the long-QT syndrome (LQT 3 ) and the Brugada syndrome. We have screened SCN5A in a large 8-generation kindred characterized by a high incidence of nocturnal sudden death, and QT-interval prolongation and the "Brugada ECG" occurring in the same subjects. An insertion of 3 nucleotides (TGA) at position 5537, predicted to cause an insertion of aspartic acid (1795insD) in the C-terminal domain of the protein, was linked to the phenotype and was identified in all electrocardiographically affected family members. ECGs were obtained from 79 adults with a defined genetic status (carriers, nϭ43; noncarriers, nϭ36). In affected individuals, PR and QRS durations and QT intervals are prolonged (PϽ0.0001 for all parameters). ST segment elevation in the right precordial leads is present as well (PϽ0.0001). Twenty-five family members died suddenly, 16 of them during the night. Expression of wild-type and mutant Na ϩ channels in Xenopus oocytes revealed that the 1795insD mutation gives rise to a 7.3-mV negative shift of the steady-state inactivation curve and an 8.1-mV positive shift of the steady-state activation curve. The functional consequence of both shifts is likely to be a reduced Na ϩ current during the upstroke of the action potential. LQT 3 and Brugada syndrome are allelic disorders but may also share a common genotype. (Circ Res. 1999;85:1206-1213.)
Na v 1.5, the pore forming a-subunit of the voltage-dependent cardiac Na + channel, is an integra... more Na v 1.5, the pore forming a-subunit of the voltage-dependent cardiac Na + channel, is an integral membrane protein involved in the initiation and conduction of action potentials. Mutations in the gene-encoding Na v 1.5, SCN5A, have been associated with a variety of arrhythmic disorders, including long QT, Brugada, and sick sinus syndromes as well as progressive cardiac conduction defect and atrial standstill. Moreover, alterations in the Na v 1.5 expression level and/or sodium current density have been frequently noticed in acquired cardiac disorders, such as heart failure. The molecular mechanisms underlying these alterations are poorly understood, but are considered essential for conception of arrhythmogenesis and the development of therapeutic strategies for prevention or treatment of arrhythmias. The unravelling of such mechanisms requires critical molecular insight into the biology of Na v 1.5 expression and function. Therefore, the aim of this review is to provide an up-to-date account of molecular determinants of normal Na v 1.5 expression and function. The parts of the Na v 1.5 life cycle that are discussed include (i) regulatory aspects of the SCN5A gene and transcript structure, (ii) the nature, molecular determinants, and functional consequences of Na v 1.5 post-translational modifications, and (iii) the role of Na v 1.5 interacting proteins in cellular trafficking. The reviewed studies have provided valuable information on how the Na v 1.5 expression level, localization, and biophysical properties are regulated, but also revealed that our understanding of the underlying mechanisms is still limited.
Biochemical and Biophysical Research Communications, 2006
Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven... more Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays >60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.
Calbindin-D 28K is suggested to play a postsynaptic role in neurotransmission and in the regulati... more Calbindin-D 28K is suggested to play a postsynaptic role in neurotransmission and in the regulation of the intracellular Ca 2+ concentration. However, it is still unclear whether calbindin-D 28K has a role in the regulation of exocytosis, either as Ca 2+ buffer or as Ca 2+ sensor. Amperometric recordings of catecholamine exocytosis from wild-type and calbindin-D 28K knockout mouse chromaffin cells reveal a strong reduction in the number of released vesicles, as well as in the amount of neurotransmitter released per fusion event in knockout cells. However, Ca 2+ current recordings and Ca 2+ imaging experiments, including video-rate confocal laser scanning microscopy, revealed that the intracellular Ca 2+ dynamics are remarkably similar in wild-type and knockout cells. The combined results demonstrate that calbindin-D 28K plays an important and dual role in exocytosis, affecting both release frequency and quantal size, apparently without strong effects on intracellular Ca 2+ dynamics. Consequently, the possibility that calbindin-D 28K functions not only as a Ca 2+ buffer but also as a modulator of vesicular catecholamine release is discussed.
Cardiac conduction defects associate with mutations in SCN5A, the gene encoding the cardiac Na ϩ ... more Cardiac conduction defects associate with mutations in SCN5A, the gene encoding the cardiac Na ϩ channel. In the present study, we characterized a family in which the proband was born in severe distress with irregular wide complex tachycardia. His older sister died at 1 year of age from severe conduction disease with similarly widened QRS-complexes. Mutational analysis of SCN5A in the proband demonstrated compound heterozygosity for a nonsense mutation (W156X), inherited from the father, and a missense mutation (R225W), inherited from the mother. Genotyping on DNA extracted from tissue from the deceased sibling revealed the same SCN5A genotype. Injection of cRNA encoding the W156X mutation in Xenopus oocytes did not produce any current. The R225W substitution neutralizes the third Arg residue within the voltage-sensing segment of domain I. Expression studies showed that this mutation leads to a severe reduction in I Na and is also associated with gating changes. Histological examination of the heart from the deceased sibling revealed changes consistent with a dilated type of cardiomyopathy and severe degenerative abnormalities of the specialized conduction system. The occurrence of compound heterozygosity for these two mutations implies that the proband carries solely severely dysfunctional cardiac Na ϩ channels. This explains his severe phenotype and that of his deceased sister who had been a carrier of the same genotype. The morphological changes within the heart of the deceased sibling may have occurred secondary to the Na ϩ channel abnormality and contributed to the severity of the disorder in this individual. (Circ Res. 2003;92:159-168.)
Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-domina... more Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant ␣-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and subcellular compartments outside the ER suggesting an effectively haplo-insufficient effect.
Kv11.1 (hERG) blockers with comparable potencies but different binding kinetics might display div... more Kv11.1 (hERG) blockers with comparable potencies but different binding kinetics might display divergent pro-arrhythmic risks. In the present study, we explored structure-kinetics relationships in four series of Kv11.1 blockers next to their structure-affinity relationships. We learned that despite dramatic differences in affinities and association rates, there were hardly any variations in the dissociation rate constants of these molecules with residence times (RTs) of a few minutes only. Hence, we synthesized sixteen novel molecules, in particular in the pyridinium class of compounds, to further address this peculiar phenomenon. We found molecules with very short RTs (e.g., 0.34 min for 37) and much longer RTs (e.g., 105 min for 38). This enabled us to construct a kon-koff-KD kinetic map for all compounds and subsequently divide the map into four provisional quadrants, providing a possible framework for a further and more precise categorization of Kv11.1 blockers. Additionally, two...
Developmental dynamics : an official publication of the American Association of Anatomists, 2005
Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 ... more Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like ...
Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fur... more Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate-(InsP 3 )-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca 2+ ] i with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca 2+ ] i oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 lM) and U73122 (10 lM) evoked similar irregular Ca 2+ responses in platelets, but in this case in the absence of InsP 3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsP 3 -induced Ca 2+ release from stores was stimulated by modest Ca 2+ concentrations, pointing to a mechanism of InsP 3 -dependent Ca 2+ -induced Ca 2+ release (CICR). This process was completely inhibitable by heparin. The Ca 2+ release by InsP 3 , but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca 2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP 3 . In contrast, U73122 caused a heparin/cAMP-insensitive Ca 2+ leak from stores that differed from those used by InsP 3 . Taken together, these results demonstrate that InsP 3 receptor channels play a crucial role in the irregular, spiking Ca 2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP 3 formation. The irregular Ca 2+ release events appear to be subjected to extensive regulation by: (a) InsP 3 level, (b) the potentiating effect of elevated Ca 2+ on InsP 3 action via CICR, (c) InsP 3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP 3 channel-independent Ca 2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca 2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca 2+ -release process.
1. Human platelets respond to agonists of G protein (G q )-coupled receptors by generating an irr... more 1. Human platelets respond to agonists of G protein (G q )-coupled receptors by generating an irregular pattern of spiking changes in cytosolic Ca 2+ ([Ca 2+ ] i ). We have investigated the ADPinduced Ca 2+ responses of single, Fluo-3-loaded platelets in the presence or absence of autologous plasma or whole blood under flow conditions.
The International Journal of Developmental Biology, 2004
The clinical application of stem cell therapies is still limited by the ability to produce define... more The clinical application of stem cell therapies is still limited by the ability to produce defined, differentiated cell populations in large numbers in culture. High throughput screens to identify factors which enhance differentiation to particular lineages and promote expansion of precursors in culture are dependent on the development of sensitive and reproducible assays for screening.
A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were... more A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, G~ was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary unitary channel conductance junctional currents was detected corresponding to an " of ~ 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic G~ voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.
Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinc... more Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinct diseases associated with sudden death: one form of the long-QT syndrome (LQT 3 ) and the Brugada syndrome. We have screened SCN5A in a large 8-generation kindred characterized by a high incidence of nocturnal sudden death, and QT-interval prolongation and the "Brugada ECG" occurring in the same subjects. An insertion of 3 nucleotides (TGA) at position 5537, predicted to cause an insertion of aspartic acid (1795insD) in the C-terminal domain of the protein, was linked to the phenotype and was identified in all electrocardiographically affected family members. ECGs were obtained from 79 adults with a defined genetic status (carriers, nϭ43; noncarriers, nϭ36). In affected individuals, PR and QRS durations and QT intervals are prolonged (PϽ0.0001 for all parameters). ST segment elevation in the right precordial leads is present as well (PϽ0.0001). Twenty-five family members died suddenly, 16 of them during the night. Expression of wild-type and mutant Na ϩ channels in Xenopus oocytes revealed that the 1795insD mutation gives rise to a 7.3-mV negative shift of the steady-state inactivation curve and an 8.1-mV positive shift of the steady-state activation curve. The functional consequence of both shifts is likely to be a reduced Na ϩ current during the upstroke of the action potential. LQT 3 and Brugada syndrome are allelic disorders but may also share a common genotype. (Circ Res. 1999;85:1206-1213.)
American Journal of Physiology Heart and Circulatory Physiology, May 1, 2001
In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchron... more In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.
Biochemical and Biophysical Research Communications, Oct 20, 2006
Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven... more Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays >60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.
ABSTRACT The main feature of gap junctions is the passage of ions and small molecules between adj... more ABSTRACT The main feature of gap junctions is the passage of ions and small molecules between adjacent cells. In the heart, for example, junctional currents between adjacent cells are elicited by the membrane potential difference when one of the cells generates an action potential, while the other cell is still at resting membrane potential, thereby allowing propagation of the action potential. Experimentally, such gap junctional currents can be directly evoked and measured by independently controlling the membrane voltage of both cells of a cell pair. This is accomplished by “dual voltage clamping” a cell-pair, a method introduced by Spray et al. (1). Using separate electrodes for voltage control and current injection, this technique allowed accurate determination of kinetics and conductance of gap junctions (1–3). At present, the dual voltage-clamp technique is usually performed with two patch pipets (4), each on one cell of a cell pair, which control voltage and measure current simultaneously (5–8).
Journal of Molecular and Cellular Cardiology, 2003
We previously described a Dutch family in which congenital cardiac conduction disorder has clinic... more We previously described a Dutch family in which congenital cardiac conduction disorder has clinically been identified. The ECG of the index patient showed a first-degree AV block associated with extensive ventricular conduction delay. Sequencing of the SCN5A locus coding for the human cardiac Na+ channel revealed a single nucleotide deletion at position 5280, resulting in a frame-shift in the sequence coding for the pore region of domain IV and a premature stop codon at the C-terminus. Wild type and mutant Na+ channel proteins were expressed in Xenopus laevis oocytes and in mammalian cells. Voltage clamp experiments demonstrated the presence of fast activating and inactivating inward currents in cells expressing the wild type channel alone or in combination with the beta1 subinut (SCN1B). In contrast, cells expressing the mutant channels did not show any activation of inward current with or without the beta1 subunit. Culturing transfected cells at 25 degrees C did not restore the Na+ channel activity of the mutant protein. Transient expression of WT and mutant Na+ channels in the form of GFP fusion proteins in COS-7 cells indicated protein expression in the cytosol. But in contrast to WT channels were not associated with the plasma membrane. The SCN5A/5280delG mutation results in the translation into non-function channel proteins that do not reach the plasma membrane. This could explain the cardiac conduction defects in patients carrying the mutation.
A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were... more A B S T R A C T The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, G~ was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary unitary channel conductance junctional currents was detected corresponding to an " of ~ 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic G~ voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.
Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinc... more Mutations in SCN5A, the gene encoding the cardiac Na ϩ channel, have been identified in 2 distinct diseases associated with sudden death: one form of the long-QT syndrome (LQT 3 ) and the Brugada syndrome. We have screened SCN5A in a large 8-generation kindred characterized by a high incidence of nocturnal sudden death, and QT-interval prolongation and the "Brugada ECG" occurring in the same subjects. An insertion of 3 nucleotides (TGA) at position 5537, predicted to cause an insertion of aspartic acid (1795insD) in the C-terminal domain of the protein, was linked to the phenotype and was identified in all electrocardiographically affected family members. ECGs were obtained from 79 adults with a defined genetic status (carriers, nϭ43; noncarriers, nϭ36). In affected individuals, PR and QRS durations and QT intervals are prolonged (PϽ0.0001 for all parameters). ST segment elevation in the right precordial leads is present as well (PϽ0.0001). Twenty-five family members died suddenly, 16 of them during the night. Expression of wild-type and mutant Na ϩ channels in Xenopus oocytes revealed that the 1795insD mutation gives rise to a 7.3-mV negative shift of the steady-state inactivation curve and an 8.1-mV positive shift of the steady-state activation curve. The functional consequence of both shifts is likely to be a reduced Na ϩ current during the upstroke of the action potential. LQT 3 and Brugada syndrome are allelic disorders but may also share a common genotype. (Circ Res. 1999;85:1206-1213.)
Na v 1.5, the pore forming a-subunit of the voltage-dependent cardiac Na + channel, is an integra... more Na v 1.5, the pore forming a-subunit of the voltage-dependent cardiac Na + channel, is an integral membrane protein involved in the initiation and conduction of action potentials. Mutations in the gene-encoding Na v 1.5, SCN5A, have been associated with a variety of arrhythmic disorders, including long QT, Brugada, and sick sinus syndromes as well as progressive cardiac conduction defect and atrial standstill. Moreover, alterations in the Na v 1.5 expression level and/or sodium current density have been frequently noticed in acquired cardiac disorders, such as heart failure. The molecular mechanisms underlying these alterations are poorly understood, but are considered essential for conception of arrhythmogenesis and the development of therapeutic strategies for prevention or treatment of arrhythmias. The unravelling of such mechanisms requires critical molecular insight into the biology of Na v 1.5 expression and function. Therefore, the aim of this review is to provide an up-to-date account of molecular determinants of normal Na v 1.5 expression and function. The parts of the Na v 1.5 life cycle that are discussed include (i) regulatory aspects of the SCN5A gene and transcript structure, (ii) the nature, molecular determinants, and functional consequences of Na v 1.5 post-translational modifications, and (iii) the role of Na v 1.5 interacting proteins in cellular trafficking. The reviewed studies have provided valuable information on how the Na v 1.5 expression level, localization, and biophysical properties are regulated, but also revealed that our understanding of the underlying mechanisms is still limited.
Biochemical and Biophysical Research Communications, 2006
Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven... more Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays >60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.
Calbindin-D 28K is suggested to play a postsynaptic role in neurotransmission and in the regulati... more Calbindin-D 28K is suggested to play a postsynaptic role in neurotransmission and in the regulation of the intracellular Ca 2+ concentration. However, it is still unclear whether calbindin-D 28K has a role in the regulation of exocytosis, either as Ca 2+ buffer or as Ca 2+ sensor. Amperometric recordings of catecholamine exocytosis from wild-type and calbindin-D 28K knockout mouse chromaffin cells reveal a strong reduction in the number of released vesicles, as well as in the amount of neurotransmitter released per fusion event in knockout cells. However, Ca 2+ current recordings and Ca 2+ imaging experiments, including video-rate confocal laser scanning microscopy, revealed that the intracellular Ca 2+ dynamics are remarkably similar in wild-type and knockout cells. The combined results demonstrate that calbindin-D 28K plays an important and dual role in exocytosis, affecting both release frequency and quantal size, apparently without strong effects on intracellular Ca 2+ dynamics. Consequently, the possibility that calbindin-D 28K functions not only as a Ca 2+ buffer but also as a modulator of vesicular catecholamine release is discussed.
Cardiac conduction defects associate with mutations in SCN5A, the gene encoding the cardiac Na ϩ ... more Cardiac conduction defects associate with mutations in SCN5A, the gene encoding the cardiac Na ϩ channel. In the present study, we characterized a family in which the proband was born in severe distress with irregular wide complex tachycardia. His older sister died at 1 year of age from severe conduction disease with similarly widened QRS-complexes. Mutational analysis of SCN5A in the proband demonstrated compound heterozygosity for a nonsense mutation (W156X), inherited from the father, and a missense mutation (R225W), inherited from the mother. Genotyping on DNA extracted from tissue from the deceased sibling revealed the same SCN5A genotype. Injection of cRNA encoding the W156X mutation in Xenopus oocytes did not produce any current. The R225W substitution neutralizes the third Arg residue within the voltage-sensing segment of domain I. Expression studies showed that this mutation leads to a severe reduction in I Na and is also associated with gating changes. Histological examination of the heart from the deceased sibling revealed changes consistent with a dilated type of cardiomyopathy and severe degenerative abnormalities of the specialized conduction system. The occurrence of compound heterozygosity for these two mutations implies that the proband carries solely severely dysfunctional cardiac Na ϩ channels. This explains his severe phenotype and that of his deceased sister who had been a carrier of the same genotype. The morphological changes within the heart of the deceased sibling may have occurred secondary to the Na ϩ channel abnormality and contributed to the severity of the disorder in this individual. (Circ Res. 2003;92:159-168.)
Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-domina... more Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant ␣-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and subcellular compartments outside the ER suggesting an effectively haplo-insufficient effect.
Kv11.1 (hERG) blockers with comparable potencies but different binding kinetics might display div... more Kv11.1 (hERG) blockers with comparable potencies but different binding kinetics might display divergent pro-arrhythmic risks. In the present study, we explored structure-kinetics relationships in four series of Kv11.1 blockers next to their structure-affinity relationships. We learned that despite dramatic differences in affinities and association rates, there were hardly any variations in the dissociation rate constants of these molecules with residence times (RTs) of a few minutes only. Hence, we synthesized sixteen novel molecules, in particular in the pyridinium class of compounds, to further address this peculiar phenomenon. We found molecules with very short RTs (e.g., 0.34 min for 37) and much longer RTs (e.g., 105 min for 38). This enabled us to construct a kon-koff-KD kinetic map for all compounds and subsequently divide the map into four provisional quadrants, providing a possible framework for a further and more precise categorization of Kv11.1 blockers. Additionally, two...
Developmental dynamics : an official publication of the American Association of Anatomists, 2005
Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 ... more Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like ...
Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fur... more Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate-(InsP 3 )-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca 2+ ] i with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca 2+ ] i oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 lM) and U73122 (10 lM) evoked similar irregular Ca 2+ responses in platelets, but in this case in the absence of InsP 3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsP 3 -induced Ca 2+ release from stores was stimulated by modest Ca 2+ concentrations, pointing to a mechanism of InsP 3 -dependent Ca 2+ -induced Ca 2+ release (CICR). This process was completely inhibitable by heparin. The Ca 2+ release by InsP 3 , but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca 2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP 3 . In contrast, U73122 caused a heparin/cAMP-insensitive Ca 2+ leak from stores that differed from those used by InsP 3 . Taken together, these results demonstrate that InsP 3 receptor channels play a crucial role in the irregular, spiking Ca 2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP 3 formation. The irregular Ca 2+ release events appear to be subjected to extensive regulation by: (a) InsP 3 level, (b) the potentiating effect of elevated Ca 2+ on InsP 3 action via CICR, (c) InsP 3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP 3 channel-independent Ca 2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca 2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca 2+ -release process.
1. Human platelets respond to agonists of G protein (G q )-coupled receptors by generating an irr... more 1. Human platelets respond to agonists of G protein (G q )-coupled receptors by generating an irregular pattern of spiking changes in cytosolic Ca 2+ ([Ca 2+ ] i ). We have investigated the ADPinduced Ca 2+ responses of single, Fluo-3-loaded platelets in the presence or absence of autologous plasma or whole blood under flow conditions.
The International Journal of Developmental Biology, 2004
The clinical application of stem cell therapies is still limited by the ability to produce define... more The clinical application of stem cell therapies is still limited by the ability to produce defined, differentiated cell populations in large numbers in culture. High throughput screens to identify factors which enhance differentiation to particular lineages and promote expansion of precursors in culture are dependent on the development of sensitive and reproducible assays for screening.
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Papers by Martin Rook