Ya. M. Shuba

Two selective filters in the calcium channel of the molluscan neuron somatic 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.

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

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.

Sodium/calcium selectivity of cloned calcium T-type channels

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 (Kd = 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+ (PCa/PNa ∼ 21) than Cav3.1 and Cav3.2 (PCa/PNa ∼ 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.

Peculiarities of Selectivity of Three Subtypes of Low-Threshold T-Type Calcium Channels

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: ICa:IBa:ISr = 1.00:0.75:1.12 and ECa ≈ EBa ≈ ESr. For channels that were formed by α1H and α1I subunits, these ratios were as follows: ICa:IBa:ISr = 1.00:1.20:1.17, ECa ≈ EBa ≈ ESr and ICa:IBa:ISr = 1.00:1.48: 1.45, ECa ≈ EBa ≈ ESr 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).

Blocking of Cloned Low-Threshold Ca2+ Channels by Neuroleptic Drugs

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.

Irina A. Vladimirova (1933–2015)

0090-2977/16/4801-0072

Involvement of Secondary Intracellular Messengers in the Mechanisms of Purinergic Inhibition of Intestinal Smooth Muscles

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.

Expression of RNA of Subunits of Low-Threshold Calcium Channels in the Laterodorsal Nucleus of the Rat Thalamus: an Ontogenetic Aspect

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.

Specific Effects of Nifedipine on Endogenous LVA Ca2+ Channel Subtypes

1 International Center for Molecular Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. Correspondence should be addressed to T. Zhelay (e-mail: zhelay@serv.biph.kiev.ua). Nifedipine, a dihydropyridine that acts as a Ca channel antagonist with a potent relaxing effect on vascular smooth muscles, has been widely recognized as a vasodilator and an antihypertensive drug in the treatment of arterial hypertension. This agent is mostly known as a specific blocker of L-type Ca channels, although a number of studies also demonstrated the capability of nifedipine to inhibit low voltage-activated (LVA, or T-type) Ca channels in some preparations. However, the mechanism of how nifedipine discriminates various LVA channel subtypes, which is evident from functional assays, remains unclear. We used isolated thalamic neurons from 14to 17-day-old rats as an object for the respective research. According to our earlier studies, these cells possess two kinetically distinct, fast and slow, LVA Ca channel subtypes; therefore, these neurons appear to be a suitable model to investigate the T-channel subtypespecific action of nifedipine. At –30 mV, nifedipine blocked the fast thalamic LVA current with IC 50 of 21.4 μM, and the level of the maximum blockade at an infinitely high concentration, A ∞ , was 79.8%. The drug was less effective with respect to the slow LVA current, inhibiting it with IC 50 = 34.4 μM and A ∞ = 55.7%. The blocking action on both currents was voltage-dependent; it decreased with depolarization for the fast current but increased for the slow current. The latter effect was accompanied by a negative shift of the steady-state current inactivation curves. The analysis of voltage and use dependence of the blockade suggests that the action of nifedipine on the currents under study includes effects on both activation and inactivation states of the channels. These data together with our previous findings support the existence of two distinct LVA Ca channel subtypes in thalamic neurons with different sensitivities to nifedipine and provide additional information to understanding the pharmacological profile of this well-known antihypertensive drug and its effects on smooth muscles.

Preferential Block of α1H Subunit of the LVA Ca2+ Channels by Nifedipine

1 International Center for Molecular Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. Correspondence should be addressed to A. Shcheglovitov (e-mail: alexsh@serv.biph.kiev.ua). There is evidence that dihydropyridines (DHP), classical blockers of the L-type Ca channels, also inhibit T-type Ca channels in several types of central and peripheral neurons and in smooth muscle cells, though with a lower potency. Thus, therapeutic effects of DHP in cardiovascular and neurological diseases may be associated with the blockade of not only L-type, but also T-type Ca channels. We provided a detailed analysis of the effects of a therapeutically widely used DHP representative, nifedipine, on the family of α1 subunits (α1G, α1H, and α1I subtypes) of the recombinant T-type Ca channels heterologously expressed in Xenopus oocytes. It was found that the α1I and α1G subunits are virtually insensitive to nifedipine even at high concentrations (IC 50 = 238 μM and 61 μM, and the level of maximum blockade, A ∞ = 47.9% and 19.6%, respectively). However, nifedipine effectively blocked α1H subunit with IC 50 = 5 μM and A ∞ = 40.8%. The voltage and state dependences of nifedipine-mediated inhibition of the above subunits were dissimilar, suggesting distinct mechanisms of interaction of the drug with various subunits. We conclude that nifedipine is an effective blocker of only the α1H T-channel subunit, and that the concentration required for the blocking action of nifedipine with respect to this channel type is comparable with that for L-type Ca channels. In addition, our data provide characteristic functional fingerprints of α1G, α1H, and α1I, which may be correlated with their in vivo counterparts.