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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.

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.

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni(2+)-binding site with critical histidine-191 (H191) within the extracellular IS3-IS4 domain of the most Ni(2+)-sensitive Cav3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni(2+) effect and its dependence on PDC remain uncertain. Here we compared Ni(2+) action on the wild-type "Ni(2+)-insensitive" Cav3.1w/t channel and Cav3.1Q172H mutant having glutamine (Q) equivalent to H191 of Cav3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni(2+) blockade of Ca(2+), Sr(2+), or Ba(2+) currents was assessed by electrophysiology. Inhibition of Cav3.1w/t by Ni(2+) conformed to two sites binding. Ni(2+) binding with high-affinity site (IC50 = 0.03-3 μM depending on PDC) produced maximal inhibition of 20-30% and was voltage-dependent, consistent with its location within the channel's pore. Most of the inhibition (70-80%) was produced by Ni(2+) binding with low-affinity site (IC50 = 240-700 μM). Q172H-mutation mainly affected low-affinity binding (IC50 = 120-160 μM). The IC50 of Ni(2+) binding with both sites in the Cav3.1w/t and Cav3.1Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca(2+), Sr(2+), or Ba(2+) with Ni(2+). We conclude that differential Ni(2+)-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni(2+), may be occupied by PDC, influencing in turn the channel's permeation.

Calcium channels were expressed inXenopus laevis oocytes by means of matrix RNA (mRNA) extracted from the cerebellum (RNAc) and forebrain (RNAfb). In these oocytes, inward barium currents,I Ba, evoked by 40 mM Ba2+ were investigated using a double microelectrode technique. Currents expressed after injection of both RNAc and RNAfb (further referred to as RNAc- and RNAfb-expressed currents) showed a voltage-dependent characteristic typical of high-threshold calcium channels of mammalian neurons. The threshold of activation was about −40 mV, the maximum amplitude was observed at +20 mV and reversal potential at +60 mV. In both groups of oocytes, no expression of low- or high-threshold calcium channels of other types was observed. Although in both cases the expression ofI Ba had similar macrokinetics, characteristics of their stationary inactivation differed. The half-inactivation potential ranged between −32 and −16 mV, and the slope factor was 28 and 16.6 mV in RNAfb- and RNAc-injected oocytes, respectively. In both cases,I Ba were insensitive to dihydropyridines; however their relation to other pharmacological agents was different. RNAfb-expressedI Ba was completely blocked by Cd2+ (K d=10 µM) and depressed up to 70% by ω-conotoxin (1 µM), being insensitive to either whole spider toxin fromAgelenopsis aperta venom or to its FTX fraction. On the contrary, RNAc-expressedI Ba was more sensitive to Cd2+ (K d=0.1 µM), stable to ω-conotoxin, and suppressed up to 75–90% by wholeA. aperta toxin in a dilution of 1:10000, and by FTX at a concentration of 0.5 µM. The findings allow us to suggest that the forebrain and cerebellum of mammals are the structures, whose mRNA differ and provide predominant expression of voltage-dependent calcium channels of N- and P-types, respectively.

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.

Xenopus laevis oocytes were injected with total mRNA obtained from the thalamo-hypothalamic complex of adult rats. In 19 out of 32 injected oocytes a Ba2+ current was expressed after 4 days which could be activated at depolarizations to -70 mV from a holding potential of -120 mV and reached a maximum value at between -30 and -20 mV. The current inactivated monoexponentially with a time constant of about 420 +/- 10 ms (n = 6); its steady state inactivation had a half value of -78 +/- 1 mV (n = 8) and a slope (k) of 11.5 +/- 3.0. These characteristics are typical of LVA (T-type) Ca2+ channels in neurones from the corresponding brain structures, except for the much slower time course of inactivation. These currents were blocked by pharmacological antagonists specific for LVA channels (amiloride, flunarizine), but remained resistant to omega-Aga-IVA and omega-Cg-toxin. These results show that LVA Ca2+ channels can be expressed in oocytes provided that the corresponding mRNA is taken from brain neurones in which they are naturally well expressed.

Ca-channel currents expressed in Xenopus oocytes by means of messenger RNA extracted from rat thalamohypothalamic complex were studied using the double microelectrode technique. Currents were recorded in Cl − -free extracellular solutions with 40 mM Ba 2+ as a charge carrier. In response to depolarizations from a very negative holding potential ( V h = −120 mV), inward Ba 2+ current activated at around −80 mV, peaked at −30 to −20 mV and reversed at +50 mV indicating that it may be transferred through the low voltage-activated calcium channels. The time-dependent inactivation of the current during prolonged depolarization to −20 mV was quite slow and followed a single exponential decay with a time-constant of 1550 ms and a maintained component constituting 30% of the maximal amplitude. The current could not be completely inactivated at any holding potential. As expected for low voltage-activated current, steady-state inactivation curve shifted towards negative potentials. It could be described by the Boltzmann equation with half inactivation potential −78 mV, slope factor 15 mV and maintained level 0.3. Expressed Ba 2+ current could be blocked by flunarizine with K d = 0.42 μM, nifedipine, K d = 10 μM, and amiloride at 500 μM concentration. Among inorganic Ca-channel blockers the most potent was La 3+ ( K d = 0.48 μM) while Cd 2+ and Ni 2+ were not very discriminative and almost 1000-fold less effective than La 3+ ( K d = 0.52 mM and K d = 0.62 mM, respectively). Our data show that messenger RNA purified from thalamohypothalamic complex induces expression in the oocytes of almost exclusively low voltage-activated calcium channels with voltage-dependent and pharmacological properties very similar to those observed for T-type calcium current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.