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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).

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.

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).

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.

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.