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The work is devoted to the study of the effect of Staphylococcus aureus peptidoglycan on the main parameters of myometrial contractions and the mechanism of this influence. The research was carried out by tensometry and calcimetry methods. The results of the experiments demonstrated that peptidoglycan modulates all the main parameters of contractions of the non-pregnant rat uterus, including increasing their amplitude and duration by 10 and 30%, respectively, mainly by prolonging the relaxation phase (56.7 ± 1.6%): myometrial band relative to quickly shrunk and relaxed for a long time. Application of peptidoglycan on freshly isolated uterine myocytes for 5 min led to a periodic increase in their intracellular content of calcium ions. Peptidoglycan enhanced myometrial contractions caused by hyperpotassium solution at a concentration of 60 mmol. Administration of 1 μmol of nifedipine, a blocker of potential-directed L-type calcium channels, suppressed spontaneous contractions enhanced by peptidoglycan. These changes in myometrium contractility of on the background of peptidoglycan action occur not only due to increased transmembrane calcium intake, but also its mobilization from the sarcoplasmic reticulum.

Пептидоглікан Золотистого стафілокока – структурний елемент бактеріальної клітинної стінки, який вивільнюється протягом життєдіяльності мікроорганізму та розноситься кровотоком по організму. Осідаючи на тропних клітинах він реалізує патогенні та імуногенні властивості. Золотистий стафілокок досить часто висівається із сечостатевої системи жінок. Відомо, що взаємодія пептидоглікану з ендотеліальними та децидуальними клітинами матки під час вагітності ініціює такі ускладнення, як прееклампсія, стимуляція скоротливості активності матки, передчасне переривання вагітності. Однак досі достеменно не вивчені механізми дії пептидоглікану на міометрій. Мета – визначити, чи впливає пептидоглікан на спонтанну скоротливість міометрія вагітних щурів за умов відсутності ендометрія, вивчити характер спонтанних скорочень міометрія вагітних щурів під дією пептидоглікану та механізми виявлених ефектів. Матеріал і методи. Досліди проводили на гладеньком’язових смужках міометрія вагітних щурів лінії Вістар. Скоротливість міометрія вивчали методом тензометрії. Результати. Результати експериментів показали, що, пептидоглікан модулює всі основні параметри спонтанних маткових скорочень за умов відсутності ендометрія. Він збільшував амплітуду та тривалість скорочень у середньому на 18,5 % та 16 %, відповідно. Водночас частота спонтанних скорочень зменшилась у середньому на 10,5 %. Ряд параметрів скорочень міометрія свідчить про збільшення їх інтенсивності під дією пептидоглікану. Крім того, змінювалось співвідношення фаз скорочень міометрія: під дією пептидоглікану спонтанні скорочення міометрія мали відносно коротку, але високоамплітудну фазу скорочення, досить тривалий тонічний компонент та повільне розслаблення. Вказані фази скорочення міометрія на тлі дії пептидоглікану збільшились у середньому на 11,26 %, 69,23 % та 44,34 % відповідно. На тлі блокаторів потенціал керованих кальцієвих каналів L-типу та ІП3-рецепторів пептидоглікан не стимулював скорочення міометрія. Висновки. Пептидоглікан має стимулювальну дію на спонтанну скоротливість міометрія шляхом впливу безпосередньо на міометрій. Модулювальний вплив пептидоглікану на скоротливість міометрія вагітних щурів пов’язаний зі збільшенням внутрішньоклітинного рівня кальцію в гладеньких міоцитах матки, що досягається завдяки посиленню трансмембранного входу кальцію та вивільненню із саркоплазматичного ретикулуму.

The ORAI family of ion channel-forming proteins in mammals includes three members, ORAI1, ORAI2 and ORAI3, encoded by homologous genes. Of these proteins the ORAI1 one received major attention as plasma membrane constituent of store-operated calcium entry (SOCE) in non-excitable cells. The functional significance of two other proteins, ORAI2 and ORAI3, is much less defined, although both of them participate to various extends in cell-specific modulation of SOCE as well as in supporting some of the store-independent calcium entry mechanisms. Calcium signaling becomes remodeled in cancer to promote cancer hallmarks - enhanced proliferation, resistance to apoptosis, motility and metastasizing. Although such remodeling commonly involves rearrangements of the whole molecular Ca

The ORAI family of ion channel-forming proteins in mammals includes three members, ORAI1, ORAI2 and ORAI3, encoded by homologous genes. Of these proteins the ORAI1 one received major attention as plasma membrane constituent of store-operated calcium entry (SOCE) in non-excitable cells. The functional significance of two other proteins, ORAI2 and ORAI3, is much less defined, although both of them participate to various extends in cell-specific modulation of SOCE as well as in supporting some of the store-independent calcium entry mechanisms. Calcium signaling becomes remodeled in cancer to promote cancer hallmarks - enhanced proliferation, resistance to apoptosis, motility and metastasizing. Although such remodeling commonly involves rearrangements of the whole molecular Ca2+-handling toolkit of the cell (Ca2+ pumps and transporters, Ca2+-binding and storage proteins, Ca2+ entry and release channels, Ca2+-dependent effectors), Ca2+ entry through Orai-based channels is especially important, as its dysregulation may contribute to several cancer hallmarks. The latter depend on the type of Ca2+-permeable channel formed by ORAI-proteins, spatiotemporal characteristics of Ca2+ signal that this channel contributes to, and the type Ca2+-dependent effector(s) targeted by this signal, all of which may be cancer-specific. By participating in global Ca2+ entry, ORAI-based SOCE may also contribute to cytosolic Ca2+ overload of cancer cells thereby playing pro-apoptotic, antineoplastic roles which can potentially be exploited for cancer treatment. This mini review examines various aspects of ORAI proteins in malignant transformation.

We studied the role of adenylate cyclase and phospholipase C in the control of ATP-induced relaxation of carbachol-evoked contraction of smooth muscles of the guinea-pig taenia coli. We showed that ATP-induced relaxation of carbachol-caused contraction is completely realized under control conditions via activation of inositol trisphosphate-sensitive (InsP3) receptors of the sarcoplasmic reticulum of smooth muscle cells (SMCs). In the case where phospholipase C was blocked, the relaxing action of ATP on smooth muscles continues to be mediated mostly by activation of InsP3 receptors, but other mechanisms begin to participate in this process. Intracellular processes are also involved in ATP-induced relaxation where the signal is transferred from purine receptors via activation of phospholipase C under conditions of parallel activation of adenylate cyclase by forskolin; these processes also include activation of InsP3 receptors of the sarcoplasmic reticulum of SMCs and some other events. After U73122-induced blocking of phospholipase C and forskolin-induced activation of adenylate cyclase, ATP-caused relaxation can completely be removed by an inhibitor of InsP3 receptors, 2-APB. This indicates that, under the above conditions, such relaxation is realized exclusively via InsP3 receptors of the sarcoplasmic reticulum of SMCs. At the same time, ATP-induced relaxation caused by activation of phospholipase C and inactivation of adenylate cyclase is also nearly completely realized with involvement of InsP3 receptors of the sarcoplasmic reticulum. However, removing the activity of phospholipase C under conditions of blocking of adenylate cyclase and InsP3 receptors of the sarcoplasmic reticulum in SMCs leads to the recovery of ATP-induced relaxation with the participation of the other intracellular processes. Therefore, two intracellular messengers, phospholipase C and adenylate cyclase, are involved in purinergic inhibition of smooth muscles. Upon their action, multiple intracellular signal pathways are triggered. The level of their participation can be influenced by the initial functional state of intestinal SMCs. These changes are always directed toward the maintenance of normal functioning of the respective organs.

We studied the peculiarities of permeability with respect to the main extracellular cations, Na+ and Ca2+, of cloned low-threshold calcium channels (LTCCs) of three subtypes, Cav3.1 (α1G), Cav3.2 (α 1H), and Cav3.3 (α1I), functionally expressed in Xenopus oocytes. In a calcium-free solution containing 100 mM Na+ and 5 mM calcium-chelating EGTA buffer (to eliminate residual concentrations of Ca2+) we observed considerable integral currents possessing the kinetics of inactivation typical of LTCCs and characterized by reversion potentials of −10 ± 1, −12 ± 1, and −18 ± 2 mV, respectively, for Cav3.1, Cav3.2, and Cav3.3 channels. The presence of Ca2+ in the extracellular solution exerted an ambiguous effect on the examined currents. On the one hand, Ca2+ effectively blocked the current of monovalent cations through cloned LTCCs (K d = 2, 10, and 18 µM for currents through channels Cav3.1, Cav3.2, and Cav3.3, respectively). On the other hand, at the concentration of 1 to 100 mM, Ca2+ itself functioned as a carrier of the inward current. Despite the fact that the calcium current reached the level of saturation in the presence of 5 mM Ca2+ in the external solution, extracellular Na+ influenced the permeability of these channels even in the presence of 10 mM Ca2+. The Cav3.3 channels were more permeable with respect to Na+ (P Ca/P Na ∼ 21) than Cav3.1 and Cav3.2 (P Ca/P Na ∼ 66). As a whole, our data indicate that cloned LTCCs form multi-ion Ca2+-selective pores, as these ions possess a high affinity for certain binding sites. Monovalent cations present together with Ca2+ in the external solution modulate the calcium permeability of these channels. Among the above-mentioned subtypes, Cav3.3 channels show the minimum selectivity with respect to Ca2+ and are most permeable for monovalent cations.

Despite the progress in studies of the properties and functions of low-threshold calcium channels (LTCCs) [1], the mechanisms of their selectivity and permeability remain unstudied in detail. We performed a comparative analysis of the selectivity of three cloned pore-forming LTCC subunits (α1G, α1H, and α1I) functionally expressed in Xenopus oocytes with respect to bivalent alkaline-earth metal cations (Ba2+, Ca2+, and Sr2+. The relative conductivities (G) of these channels were determined according to the amplitudes of macroscopic currents (I) and potentials of zero currents (E). The currents were recorded after preliminary intracellular injection of a fast calcium buffer, BAPTA, in order to suppress the endogenous calcium-dependent chloride conductivity. Channels formed by α1G subunits demonstrated the following ratios of the amplitudes of macroscopic currents and potentials of zero current: I Ca:I Ba:I Sr = 1.00:0.75:1.12 and E Ca ≈ E Ba ≈ E Sr. For channels that were formed by α1H and α1I subunits, these ratios were as follows: I Ca:I Ba:I Sr = 1.00:1.20:1.17, E Ca ≈ E Ba ≈ E Sr and I Ca:I Ba:I Sr = 1.00:1.48: 1.45, E Ca ≈ E Ba ≈ E Sr respectively. The different macroscopic conductivities and similar potentials of zero current typical of α1G and α1I channels indicate that, probably, various bivalent cations can in a differential manner influence the stochastic parameters of functioning of these channels. At the same time, channels formed by α1H subunits are characterized by more positive potentials of zero current for Ca2+. It seems possible that the selectivity of the above channels is determined by mechanisms that mediate the selectivity of most high-threshold calcium channels (more affine binding of Ca2+ inside the pore).

It was shown earlier that low-threshold calcium current in neurons of the laterodorsal nucleus of the rat thalamus is of a multicomponent nature, and, moreover, its composition changes in the course of postnatal development of the animal. These findings raised a question on the ontogenetic development of expression of the pore-forming subunits of low voltage-activated (LVA) calcium channels (or T-type channels). At present, three isoforms of such subunits, α 1G, α1I, and α1H, have been cloned. In our study, we examined expression of mRNA of three subunits of T-type calcium channels in the laterodorsal thalamic nucleus of 1-, 5-, 15-, and 90-day (3-month)-old Wistar rats using reverse transcription and the polymerase chain reaction (PCR). Analysis of amplificates showed that mostly RNAs coding α1G and α1I subunits are expressed in the laterodorsal nucleus of rats of all age groups, while levels of expression RNAs of α1H and α1E subunits (the latter subunit of calcium channels of R type is close in its properties to subunits of LVA channels) are considerably lower (16 or 32 times smaller than the respective values for α1G and α1I subunits). We could not find a significant dependence of the expression of subunit RNAs of the above-mentioned calcium channels on the phases of ontogenetic development. It is possible to hypothesize that the age dynamics of the recorded calcium current in neurons of the laterodorsal thalamic nucleus is not related to increases in the transcription of the corresponding genes but results from post-translational changes in α1G and α1I subunits of proteins of LVA calcium channels or age-related peculiarities of regulation.

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca2+ channels functionally expressed in Xenopus oocytes and formed by three different subunits (α1G, α1H, and α1I). The α1G and α1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC50 = 98 and 243 μM, maximum blocking intensity Amax = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the α1H-subunit (IC50 = 5 μM and Amax = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of α1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of α1H- and α1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca2+ channels in native cells.

We studied the blocking action of neuroleptic drugs, haloperidol, pimozide, and fluspirilene, on three types of cloned low voltage-activated (T-type) Ca2+ channels, α1G, α1H, and α1I, functionally expressed in Xenopus oocytes. Fluspirilene and pimozide (members of the diphenylbutylpiperidine group) and haloperidol (belonging to butyrophenones) inhibited Ca2+ currents with different values of the Kd constant and maximum intensity of blocking. Effects of the neuroleptics were voltage-dependent and were accompanied by slowing-down of the kinetics of the currents. The mechanism of blocking is probably based on interaction between the neuroleptics and the channels in the activated and inactivated states. The difference in efficiency and specificity of blockade of various T-channel subtypes by neuroleptics should be considered when estimating the therapeutic significance of the tested pharmacological agents.

The activity of voltage-operated calcium channels is of primary importance for integration of the signals conducted along neuronal processes and transsynaptic transmission of messages carried by them to other neurons. The structure and function of various types of calcium channels has been the object of numerous investigations, including those carried out for many years in our Institute. This lecture briefly summarizes the main data in this field, with special attention towards recent possibilities of specific pharmacological intervention into the activity of different types of calcium channels, which is of the highest importance for effective treatment of various abnormalities in the functioning of the nervous system.

Low voltage-activated (LVA) Ca2+ conductances were characterized in the neurons of the associative laterodorsal (LD) thalamic nucleus in rat brain slices and in enzymatically isolated thalamic units using electrophysiological techniques. Voltage dependence, kinetics of inactivation, pharmacology, and selectivity of the LVA current in the thalamic neurons from animals older than 14 postnatal days were consistent with the existence of two, “fast” and “slow,” subtypes of LVA Ca2+ channels. “Slow” LVA current in enzymatically isolated thalamic neurons was much less prominent, compared with that in slice neurons, suggesting that respective channels are predominatly located on the distal dendrites. “Fast” Ca2+ channels were sensitive to nifedipine (K d−2.6 μM) and La3+ (K d−1.0 mM), whereas “slow” Ca2+ channels were sensitive to Ni2+ (25 μM). Selectivity of the “fast” Ca2+ channels was similar to that found for the LVA Ca2+ channels in other preparations (I Ca:I Sr:I Ba−1.0: 1.23: 0.94), while selectivity of the “slow” Ca2+ channels more resembled selectivity of the HVA Ca2+ channels (I Ca:I Sr:I Ba−1.0: 2.5: 3.4).

The plasmalemmal Na+−Ca2+ exchanger is a coupled Na+ and Ca2+ transport mechanism that plays an important role in regulation of Ca2+ homeoslasis in many cell types. A robust Na+−Ca2+ exchange system is present in the heart where it comprises essential Ca2+ extrusion, as well as Ca2+ entry pathways, that significantly contribute to the maintenance of cardiac contractility. The review examines the basic properties of Na+−Ca2+ exchange, the patterns of its regulation, as well as the latest achievements in the cloning and structure-function studies of a Na+−Ca2+ exchanger molecule.

Rat forebrain- and heart-derived mRNA were used to express Ca2+ channels inXenopus oocytes to study their cAMP-dependent regulation. Forebrain and heart mRNA-directed Ca2+ channel currents (I Ba, 40 mM Ba2+ were used as a charge carrier) showed similar voltage dependence and macroscopic kinetics but different pharmacology, which allowed us to attribute them to N- and L-type, respectively. Brain mRNA-directedI Ba was insensitive to the dihydropyridine (DHP) antagonist nitrendipine and the agonist Bay K 8644, but could be inhibited by 70% by 1 μM of ω-conotoxin GVIA, whileI Ba directed by cardiac mRNA was extremely sensitive to DHP. Neither forebrain, nor heart mRNA-directedI Ba could be augmented by the external applications of the β-agonist isoproterenol (ISO, 10 μM), the adenylate cyclase (AC) activator forskolin (FSK, 10 μM), the phosphodiesterase inhibitor IBMX (200 μM), or their mixtures. “Cardiac”I Ba was also unresponsive to the external applications of a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (500 μM), as well as to the direct intracellular infusion of cAMP (300 μM). Blockade of cAMP-dependent phosphorylation pathway by intracellular perfusion of the oocytes with 200 μM Rp-cAMP plus 200 μM of a synthetic protein kinase A (PKA) inhibitor peptide also exerted no effect on the basal level ofI Ba, suggesting that the expressed Ca2+ channels are not fully phosphorylated in the resting state. Measurements of the concentration of cAMP in the control and heart mRNA-injected oocytes, using an enzyme-immunoassay system, showed that they display a similar basal cAMP concentration (2.0–2.5 μM); however, application of ISO + FSK increased the cAMP concentration 2- to 3-fold in mRNA-injected oocytes, but not in control oocytes. Thus, our data demonstrate that injection of rat cardiac mRNA intoXenopus oocytes results in the expression of receptor-stimulated AC and L-type Ca2+ channels, which do not respond to cAMP or PKA inhibitors. Unresponsiveness to cAMP-dependent regulation is not channel type-specific, since N-type Ca2+ channels expressed by means of forebrain mRNA are also insensitive to such regulation. Unresponsiveness of the channels to cAMP-mediated regulation is most probably due to lack/inaccessibility of PKA-dependent phosphorylation site(s), or loss of functional significance of phosphorylation.

Calcium channels were expressed inXenopus oocytes by means of messenger RNA extracted from the rat thalamo-hypothalamic complex, mRNA(h). Inward barium currents,I Ba, were recorded in Cl−-free extracellular solution with 40 mM Ba2+ as a charge carrier, using two-microelectrode technique. Depolarizations from a very negative holding potential (V h=−120 mV) began to activateI Ba at about −80 mV; this current peaked at −30 to −20 mV and reversed at +50 mV, indicating that I Ba may be transferred through the low voltage-activated (LVA) calcium channels. The time-dependent inactivation of the current during a prolonged depolarization to −20 mV was quite slow, followed a single exponential decay with a time constant of 1550 msec, and contained a residual component constituting 30% of the maximum amplitude. The current could not be completely inactivated at any holding potential. As expected for LVA current, a steady-state inactivation curve was shifted towards negative potentials. It could be described by the Boltzmann's equation with the half-inactivation potential of −78 mV, slope factor of 15 mV, and residual level of 0.3. ExpressedI Ba could be blocked by flunarizine (K d=0.42 µM), nifedipine (K d=10 µM), and amiloride at a 500 µM concentration. Among the inorganic Ca2+ channel blockers, the most potent was La3+ (K d=0.48 µM), while Cd2+ and Ni2+ were not very selective and almost thousand-fold less effective (K d=0.52 mM andK d=0.62 mM, respectively) than La3+. Our data show that mRNA(h) induces expression in the oocytes of almost exclusively LVA Ca2+ channels with voltage-dependent and pharmacological properties very similar to those observed for T-type Ca2+ current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.

Expression of voltage-gated calcium and sodium ionic channels was found with the help of electrophysiological methods in the membrane of oocytes of theXenopus laevis frog following the injection of total RNA from the brain of 15-to-20-day-old rats. The amplitudes of currents through these channels were much higher than those induced by poly(A)+-mRNA from the same source. Barium currents induced by both RNA preparations were insensitive to Bay K 8644 (10 µM) and nitrendipine (50 µM), but were blocked by addition of 100 µM Cd2+ to extracellular solution; in both cases ω-conotoxin GVIA (1 µM) suppressed currents through the expressed calcium channels. The conclusion is that processes responsible for the expression of alien ionic channels in oocyte membrane become more intensive following the injection of total RNA separated in a sucrose gradient than those following the injection of poly(A)+-mRNA. The results suggest that the effectiveness of the expression is positively affected by some factors contained in the preparations of total RNA. The question of the nature of these factors remains open.

The expression of two types of voltage-gated ion channels of the inflowing current ("fast" sodium channels, sensitive to tetrodotoxin, and high-threshold calcium channels) was detected by electrophysiological methods in the membrane ofXenopus oocytes, after injection of poly(A)+-mRNA from the brains of 18- to 20-day-old rats. When Cd2+ (200 µmoles/liter) was added to the extracellular solution, the barium current through the expressed calcium channels was completely suppressed, but no sensitivity to D-600 (20 µmoles/liter) and nitrendipine (50 µmoles/liter) was exhibited. A peptide blocker of the high-threshold calcium channels of the neuron membrane, ω-conotoxin GVIA, in a concentration of 1 µmole/liter led to 20–40 min suppression of the barium current expressed in the oocyte. Steady-state inactivation of this current could be described by the Boltzman formula, using the values of the half-inactivation potential V1/2=−50 mV and the steepness factor k=14 mV. It is concluded that in potential-dependent and pharmacological properties, the calcium channels expressed in the oocyte, despite the absence of any appreciable time-dependent inactivation, most resemble the high-threshold inactivatable (HTI- or N-type) calcium channels of the neuron membrane.

Modification of the calcium channel of the somatic membrane of molluscan neurons under the influence of EDTA and other Ca-binding agents was investigated. The results showed that there are two selective filters in the calcium channel of this membrane. The first is located near the outer pore of the calcium channel and it binds bivalent cations in the order:pKCa:pKSr:pKBa:pKMg=6.6:5.5:4.8:4.2. This external filter regulates selectivity of the channel relative to the charge of the cation and it conjecturally contains several carboxyl groups. The second selective filter lies inside the channel and regulates permeability for ions with a single charge. It is suggested that the structure of the inner filter closely resembles that postulated by Hille for the selective filter of the sodium channel, and that it contains one carboxyl group. The results of investigation of the effect of Ca++, Cd++, and H+ on the fast sodium current of the somatic membrane showed that it is not blocked by these ions, but the decrease observed in its amplitude is connected with a change in the membrane surface potential and a corresponding change in the juxtamembranous concentration of carrier ions. On the basis of the experimental results it is postulated that the selective filter of the fast sodium channel of the molluscan neuron somatic membrane does not contain a carboxyl group.

Using a refined patch clamp technique, a study was made of single calcium channels of spinal ganglia neurons on a cell-attached membrane site in newborn rats; these convey the basic (high threshold) component of calcium current. Findings show that currents carried by calcium ions at a concentration of 60 mM in the recording pipet changes from 0.58±0.05 to 0.43±0.05 pA with a change in potential of 20 mV. This corresponds to a channel conductance of 7±0.5 pS. The distribution of open time was monoexponential with a time constant of about 0.75 msec, independent of membrane potential. Distribution of closed time approached a biexponential time course. The fast component (0.8 msec) was voltage-dependent, while the slow component decreased from 22 to 4 msec when depolarization increased by 20 mV. Using experimentally obtained time parameters which describe single calcium channel function, and assuming a three-tier model of the channel, the numerical values of the constants of transition rates between individual states were determined.

Three types of electrically-operated calcium channels have been identified in the membrane of cultured dorsal root ganglion neurons from mouse embryos using patch clamp technique. Low-threshold inactivating (LTI) channels with the lowest unitary conductance (5.7 pS with 60 mM Sr2+ or 7.2 pS with 60 mM Ba2+) preserved their activity for a long time on excised membrane patches and were insensitive to dihydropyridine Ca channel agonist Bay K8644. Corresponding whole-cell current could be decreased by 40% with 25 microM D-600. High-threshold inactivating (HTI) channels had somewhat higher unitary conductance (7 pS and 11.4 pS for Sr2+ or Ba2+ at 60 mM concentration) and were much more dependent upon intracellular metabolic support. D-600 inhibited the corresponding HTI whole-cell current by about 10%. High-threshold non-inactivating (HTN) channels were the most conducting ones (9 pS and 18.4 pS for 60 mM Sr2+ or 60 mM Ba2+) and their functioning was strongly metabolic dependent. Whole-cell HTN current could be slightly enhanced by 10 microM Bay K8644 due to some prolongation of channel mean open time. Effect of Bay K8644 was much less pronounced than that reported for cardiac cells. HTN whole-cell current could be almost completely blocked by 25 microM D-600. The described three types of calcium channels revealed different potential dependence and absolute values of mean channel open time.

The kinetics of the slow current carried by sodium ions through potential-dependent calcium channels after addition of EDTA to calcium-free external solution was investigated in experiments by the intracellular dialysis method on isolatedHelix pomatia neurons. The activation kinetics of this current was similar to that of the calcium current and could be described by the use of the square of the activation variable m in Hodgkin-Huxley equations. The decay (inactivation) kinetics of the induced sodium current during prolonged depolarization is biexponential in character. It is suggested that decay of the sodium currents takes place as a result of two independent processes: potential-dependent inactivation with a time constant τh∼1 sec, taking place as far as a certain steady-state level h∞, and a decrease in current connected with Na+ accumulation inside the cell during passage of the current and a consequent change in the sodium electrochemical potential (τc∼10 sec). It is concluded that modification of the calcium channels, so that they acquire the ability to conduct sodium, has no significant effect on the gating mechanisms responsible for opening and closing of the channels.

The characteristics of slow inward sodium currents arising in response to membrane depolarization were studied in experiments on isolated dialyzed neurons of the snailHelix pomatia when the calcium-chelating agent EDTA was added to the calcium-free external solution. Values of the relative permeability of the corresponding ionic channels, determined from the shift of the equilibrium potential, were: PNa+:PLi+: $$P_{NH_2 NH_3 } + :P_{OHNH_3 } $$ +=1.00:0.80:0.55:0.21. The ratio between these values for "fast" sodium channels was 1.00:1.04:0.44:0.19. The induced sodium current was blocked by D-600 and nifedipine, which block calcium channels, more effectively than the calcium current of the same membrane (the corresponding dissociation constants were 10−5 and 0.8·10−5 mole/liter for the induced sodium current compared with 2.6·10−5 and 2.3·10−5 mole/liter for the calcium current). It is postulated on the basis of these data that the calcium channels have a principal selective filter similar to that of sodium channels, but also an additional binding site for bivalent cations, which prevents entry of monovalent cations into the channel. The addition of calcium-chelating agents to the calcium-free external solution liberates this site and thereby modifies the calcium channel into a sodium channel.

Activation kinetics of single high-threshold inactivating (HTI orN-type) calcium channels of cultured dorsal root ganglion cells from mouse embryos was studied using a patchclamp method. Calcium channels displayed bursting activity. The open-time histogram was single exponential with an almost potential-independent mean open time τop. The closed-time histogram was multicomponent; at least three of the components were associated with the activation process. The “fast” exponential component with the potential-independent time constant τ cl included all intraburst gaps, while two “slower” ones with potential-dependent time constants τ cl described shut times between bursts and between clusters of bursts. The burst length histogram was biexponential. The “fast” component with a relatively potential-independent time constant τ bur described short, isolated channel openings while the “slow” component characterized real bursts with a potential-dependent mean life time. The waiting-time histogram could be fitted by a difference of two exponentials with time constants being the same as τ cl and τ cl . The data obtained were described in the frame of a 4-state sequential model of calcium channel activation, in which the first two stages are formally attributed to potential-dependent transmembrane transfer of two charged gating particles accompanying the channel transitions between three closed states, and the third one to fast conformational changes in channel protein leading to the opening of the channel. The rate constants for all transitions were defined. The validity of the proposed model for both low-threshold inactivating (LTI orT-type) and high-threshold noninactivating (HTN orL-type) calcium channels is discussed.

It is clear now that the calcium conductance of neuronal membrane is based on the activity of several types of calcium channels. Originally two types of calcium channels were discovered in the somatic membrane of DRG neurons of rat (Veselovsky and Fedulova 1983). As they are different in their operational potential range, these calcium channels can be identified as low- and high-threshold. Channels of the first type become active at membrane potentials between -60 and -30 mV and reveal a clear voltage- and time-dependent inactivation (L TI channels, or T type according to Tsien‘s nomenclature). The second type required more positive potentials for activation (up to 0 m V) and their inactivation was much less pronounced. Subsequently this finding was confirmed in a number of investigations in which properties of both types of channels were studied in detail (Carbone and Lux, 1984a, b, 1987 a, b; Fedulova et al., 1985; Kostyuk et al., 1986). However, it has been shown recently with sensory neurons of vertebrates that high-threshold calcium channels are also nonhomogeneous and can be separated into two classes - inactivating and noninactivating (HTI, HTN; Nand L according to Tsien) (Nowycky et al., 1985b; Kostyuk et al., 1987; Fox et al., 1987 a, b).