Voltage and Ca2+ activation of single large-conductance Ca2+-activated K+ channels described by a two-tiered allosteric gating mechanism

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, q(eff), of 2.3 +/- 0.6 e(o)). Estimates of q(eff) were little changed for intracellular Ca2+ (Ca2+(i)) ranging from 0.0003 to 1,024 microM. Increasing Ca2+(i) from 0.03 to 1,024 microM shifted the voltage for half maximal activation (V(1/2)) 175 mV in the hyperpolarizing direction. V(1/2) was independent of Ca2+(i) for Ca2+(i) < or = 0.03 microM, indicating that the channel can be activated in the absence of Ca2+(i). Open and closed dwell-time distributions for data obtained at different Ca2+(i) and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (q(eff) = -0.5 e(o)) and an increase in the mean opening rate (q(eff) = 1.8 e(o)), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+(i) (approximately 0 through 1,024 microM), voltage (+80 to -80 mV), and Po (10(-4) to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.     

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.     

Atorvastatin inhibits angiotensin II-induced T-type Ca2+ channel expression in endothelial cells

Ca2+ channels are involved in the regulation of vascular functions. Angiotensin II is implicated in the development of atherosclerosis and vascular remodeling. In this study, we demonstrated that angiotensin II preferentially increased the expression of alpha1G, a T-type Ca2+ channel subunit, via AT1 receptors in endothelial cells. Angiotensin II-induced expression of alpha1G was inhibited by pretreatment with atorvastatin and the MEK1/2 inhibitor, PD98059. The effect of atorvastatin was reversed by mevalonate and farnesyl pyrophosphate which implicates the activation of the small GTP-binding protein, Ras. Our data indicate that angiotensin II induces alpha1G expression in endothelial cells via AT1 receptors, Ras and MEK. Angiotensin II-induced migration of endothelial cells in a wound healing model was inhibited by incubation with mibefradil, a T-type Ca2+ channel blocker. Our data indicate that angiotensin II induces T-type Ca2+ channels in endothelial cells, which may play a role in the development of vascular disorders.     

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells

Hydrogen sulfide (H₂S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H₂S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Ca_v3.2 T-type Ca²⁺ channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Ca_v3.2. In the present study, we asked if endogenous and exogenous H₂S participates in regulation of the channel functions in Ca_v3.2-transfected HEK293 (Ca_v3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Ca_v3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5 mM did not affect T-currents in Ca_v3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5 mM, but not 0.1–0.3 mM, increased T-currents in Ca_v3.2-HEK293 cells. Similarly, Na₂S, another H₂S donor, at 0.1–0.3 mM significantly increased T-currents in the presence, but not absence, of PPG in Ca_v3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na₂S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na₂S was higher than NaHS. These results suggest that the function of Ca_v3.2 T-channels is tonically enhanced by endogenous H₂S synthesized by CSE in Ca_v3.2-HEK293 cells, and that exogenous H₂S is capable of enhancing Ca_v3.2 function when endogenous H₂S production by CSE is inhibited. In addition, Na₂S is considered a more potent H₂S donor than NaHS in vitro as well as in vivo.     

TRPC1 protein forms only one type of native store-operated channels in HEK293 cells

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I_max channels, while the properties of I_min and I_NS channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current–voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I_max channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I_max channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I_max channels but is not an essential subunit for other store-operated channel types in HEK293 cells.     

Modulation of T-type Ca2+ channels by corticotropin-releasing factor through protein kinase C pathway in MN9D dopaminergic cells

Corticotrophin-releasing factor (CRF) is the main regulator of the body's stress axis and its signal is translated through G-protein-coupled CRF receptors (CRF-R1, CRF-R2). Even though CRF receptors are present in the midbrain dopamine neurons, the cellular mechanism of CRF action is not clear yet. Since voltage-dependent Ca(2+) channels are highly expressed and important in dopamine neuronal functions, we tested the effect of CRF on voltage-dependent Ca(2+) channels in MN9D cells, a model of dopamine neurons. The application of CRF-related peptide, urocortin 1, reversibly inhibited T-type Ca(2+) currents, which was a major Ca(2+) channel in the cells. The effect of urocortin was abolished by specific CRF-R1 antagonist and was mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate. PKC inhibitors abolished the effect of urocortin. These results suggest that urocortin modulates T-type Ca(2+) channel by interacting with CRF-R1 via the activation of PKC signal pathway in MN9D cells.     

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.     

Effects of intracellular magnesium on Kv1.5 and Kv2.1 potassium channels

We characterized the effects of intracellular Mg²⁺ (Mg²⁺_i) on potassium currents mediated by the Kv1.5 and Kv2.1 channels expressed in Xenopus oocytes. Increase in Mg²⁺_i caused a voltage-dependent block of the current amplitude, apparent acceleration of the current kinetics (explained by a corresponding shift in the steady-state activation) and leftward shifts in activation and inactivation dependencies for both channels. The voltage-dependent block was more potent for Kv2.1 [dissociation constant at 0 mV, K_d(0), was ~70 mM and the electric distance of the Mg²⁺ binding site, , was 0.2] than for the Kv1.5 channel [K_d(0)~40 mM and =0.1]. Similar shifts in the voltage-dependent parameters for both channels were described by the Gouy-Chapman formalism with the negative charge density of 1 e^–/100 Ų. Additionally, Mg²⁺_i selectively reduced a non-inactivating current and increased the accumulation of inactivation of the Kv1.5, but not the Kv2.1 channel. A potential functional role of the differential effects of Mg²⁺_i on the Kv channels is discussed.     

Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Expression of polycystin-1 C-terminal fragment enhances the ATP-induced Ca2+ release in human kidney cells

Polycystin-1 (PC1) is a membrane protein expressed in tubular epithelia of developing kidneys and in other ductal structures. Recent studies indicate this protein to be putatively important in regulating intracellular Ca(2+) levels in various cell types, but little evidence exists for kidney epithelial cells. Here we examined the role of the PC1 cytoplasmic tail on the activity of store operated Ca(2+) channels in human kidney epithelial HEK-293 cell line. Cells were transiently transfected with chimeric proteins containing 1-226 or 26-226 aa of the PC1 cytoplasmic tail fused to the transmembrane domain of the human Trk-A receptor: TrkPC1 wild-type and control Trk truncated peptides were expressed at comparable levels and localized at the plasma membrane. Ca(2+) measurements were performed in cells co-transfected with PC1 chimeras and the cytoplasmic Ca(2+)-sensitive photoprotein aequorin, upon activation of the phosphoinositide pathway by ATP, that, via purinoceptors, is coupled to the release of Ca(2+) from intracellular stores. The expression of TrkPC1 peptide, but not of its truncated form, enhanced the ATP-evoked cytosolic Ca(2+) concentrations. When Ca(2+) assays were performed in HeLa cells characterized by Ca(2+) stores greater than those of HEK-293 cells, the histamine-evoked cytosolic Ca(2+) increase was enhanced by TrkPC1 expression, even in absence of external Ca(2+). These observations indicate that the C-terminal tail of PC1 in kidney and other epithelial cells upregulates a Ca(2+) channel activity also involved in the release of intracellular stores.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

Proportions of Ca2+ Channel Subtypes in Chick or Rat P2 Fraction and NG108-15 Cells Using Various Ca2+ Blockers

The proportions of calcium (Ca²⁺) channel subtypes in chick or rat P₂ fraction and NG 108-15 cells were investigated using selective L-, N-, P- and P/Q- type Ca²⁺ channel blockers. KCl-stimulated ⁴⁵Ca²⁺ uptake by chick P₂ fraction was blocked by 40~50% using N-type Ca²⁺ channel blockers [-conotoxin GVIA, aminoglycoside antibiotics and dynorphin A(1–13)], but was not inhibited by P- or P/Q-type blockers (-agatoxin IVA or -conotoxin MVIIC). On the other hand, KCl-stimulated ⁴⁵Ca²⁺ uptake by rat P₂ fraction was blocked by 30~40% using P- or P/Q-type Ca²⁺ channel blockers, but was not inhibited by N-type Ca²⁺ channel blockers. The L-type Ca²⁺ channel blockers 1,4-dihydropyridines, diltiazem and verapamil, but not calciseptine (CaS), inhibited both KCl-stimulated ⁴⁵Ca²⁺ uptake and veratridine-induced ²²Na⁺ uptake by chick or rat P₂ fraction with similar IC₅₀ values. CaS did not have any effect on ⁴⁵Ca²⁺ uptake by either chick or rat P₂ fraction. In NG108-15 cells, CaS, -agatoxin IVA and -conotoxin MVIIC, but not -conotoxin GVIA, inhibited KCl-stimulated ⁴⁵Ca²⁺ uptake by 30–40%. Various combinations of these Ca²⁺ channel blockers had no significant additional effects in chick or rat P₂ fraction or NG 108-15 cells. These findings suggest that KCl-stimulated ⁴⁵Ca²⁺ uptake by chick or rat P₂ fraction and NG 108-15 cells is a convenient and useful model for screening whether or not natural or synthetic substances have selective effects as L-, N-, P-, or P/Q- type Ca²⁺ channel antagonists or agonists.     

Pertussis toxin-sensitive pathway inhibits glucose-stimulated Ca2+ signals of rat islet beta-cells by affecting L-type Ca2+ channels and voltage-dependent K+ channels

A role of pertussis toxin (PTX)-sensitive pathway in regulation of glucose-stimulated Ca2+ signaling in rat islet beta-cells was investigated by using clonidine as a selective agonist to alpha2-adrenoceptors which link to the pathway. An elevation of extracellular glucose concentration from 5.5 to 22.2 mM (glucose stimulation) increased the levels of [Ca2+]i of beta-cells, and clonidine reversibly reduced the elevated levels of [Ca2+]i. This clonidine effect was antagonized by yohimbine, and abolished in beta-cells pre-treated with PTX. Clonidine showed little effect on membrane currents including those through ATP-sensitive K+ channels induced by voltage ramps from -90 to -50 mV. Clonidine showed little effect on the magnitude of whole-cell currents through L-type Ca2+ channels (ICa(L)), but increased the inactivation process of the currents. Clonidine increased the magnitude of the voltage-dependent K+ currents (IVK). These clonidine effects on ICa(L) and IVK were abolished in beta-cells treated with PTX or GDP-betaS. These results suggest that the PTX-sensitive pathway increases IVK activity and decreases ICa(L) activity of islet beta-cells, resulting in a decrease in the levels of [Ca2+]i elevated by depolarization-induced Ca2+ entry. This mechanism seems responsible at least in part for well-known inhibitory action of PTX-sensitive pathway on glucose-stimulated insulin secretion from islet beta-cells.     

Oscillations of cytoplasmic concentrations of Ca2+ and K+ in fused L cells

Summary Using Ca²⁺- and K⁺-selective microelectrodes, the cytosolic free Ca²⁺ and K⁺ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca²⁺ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca²⁺ concentration were observed in the submicromolar ranges. During the Ca²⁺ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca²⁺ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca²⁺ concentration down to 10^–7 m or by applying a Ca²⁺ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca²⁺-activated K⁺ channel, sizable Ca²⁺ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K⁺ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K⁺ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca²⁺ level, which, in turn, periodically stimulate Ca²⁺-activated K⁺ channels.     

Selectivity signatures of three isoforms of recombinant T-type Ca channels

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ (K_D1 ∼ 6 μM) and for Ca²⁺ current saturation (K_D2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.