T-type current modulation by the actin-binding protein Kelch-like 1.

We report a novel form of modulation of T-type calcium currents carried out by the neuronal actin-binding protein (ABP) Kelch-like 1 (KLHL1). KLHL1 is a constitutive neuronal ABP localized to the soma and dendritic arbors; its genetic elimination in Purkinje neurons leads to dendritic atrophy and motor insufficiency. KLHL1 participates in neurite outgrowth and upregulates voltage-gated P/Q-type calcium channel function; here we investigated KLHL1's role as a modulator of low-voltage-gated calcium channels and determined the molecular mechanism of this modulation with electrophysiology and biochemistry. Coexpression of KLHL1 with Ca(V)3.1 or Ca(V)3.2 (alpha(1G) or alpha(1H) subunits) caused increases in T-type current density (35%) and calcium influx (75-83%) when carried out by alpha(1H) but not by alpha(1G). The association between KLHL1 and alpha(1H) was determined by immunoprecipitation and immunolocalization in brain membrane fractions and in vitro in HEK-293 cells. Noise analysis showed that neither alpha(1H) single-channel conductance nor open probability was altered by KLHL1, yet a significant increase in channel number was detected and further corroborated by Western blot analysis. KLHL1 also induced an increase in alpha(1H) current deactivation time (tau(deactivation)). Interestingly, the majority of KLHL1's effects were eliminated when the actin-binding motif (kelch) was removed, with the exception of the calcium influx increase during action potentials, indicating that KLHL1 interacts with alpha(1H) and actin and selectively regulates alpha(1H) function by increasing the number of alpha(1H) channels. This constitutes a novel regulatory mechanism of T-type calcium currents and supports the role of KLHL1 in the modulation of cellular excitability.

Kelch-like 1 protein upregulates T-type currents by an actin-F dependent increase in α(1H) channels via the recycling endosome.

The neuronal protein Kelch-like 1 (KLHL1) is a novel actin-binding protein that modulates neuronal structure and function. KLHL1 knockout mice exhibit dendritic atrophy in cerebellar Purkinje neurons and motor dysfunction. Interestingly, KLHL1 upregulates high and low voltage-gated calcium currents (Ca(V)2.1 and Ca(V)3.2) and interacts with their respective principal subunits, α(1A) and α(1H). We reported the mechanism of enhanced Ca(V)3.2 (α(1H)) current density (and calcium influx) by KLHL1 is due to an increase in channel number (N) that requires the binding of actin. In this report we further elucidate the role of the actin cytoskeleton in this process using pharmacological tools to disrupt or stabilize actin filaments and to prevent protein trafficking and vesicle recycling. Disruption of the cytoskeleton did not affect the basal activity of α(1H), but did eliminate its modulation by KLHL1. In contrast, actin-F stabilization on its own increased basal α(1H) activity similar to KLHL1 but without synergy in its presence, suggesting KLHL1 requires actin-polymerization to increase α(1H) currents. Noise analysis revealed that actin polymerization induced an increase in N and P(o), in contrast to increased N in the presence of KLHL1. Interestingly, pharmacological or genetic disruption of endosomal recycling eliminated the increase in channel number by KLHL1 demonstrating this effect occurs via enhanced α(1H) re-insertion through the recycling endosome. Our findings afford insight on a novel mechanism of T-type channel modulation that could have overall functional implications for T-type channel function in the brain.

Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart.

During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca(2+)) channels are critical for maintaining both contractility and excitability. In the present study, we identified the Ca(V)3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. Ca(V)3.1d is a splice variant of the alpha1G, T-type Ca(2+) channel. Immunohistochemical localization showed expression of alpha1G Ca(2+) channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the alpha1G channels. Dihydropyridine-resistant inward Ba(2+) or Ca(2+) currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the alpha1G channel expressed in human embryonic kidney-293 cells. Ni(2+) blockade discriminates among T-type Ca(2+) channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca(2+) handling proteins. We demonstrate that 100 micromol/L Ni(2+) partially blocked alpha1G currents under physiological external Ca(2+). We conclude that alpha1G T-type Ca(2+) channels are functional in midgestational fetal myocardium.

Mibefradil block of cloned T-type calcium channels.

Mibefradil is a tetralol derivative chemically distinct from other calcium channel antagonists. It is a very effective antihypertensive agent that is thought to achieve its action via a higher affinity block for low-voltage-activated (T) than for high-voltage-activated (L) calcium channels. Estimates of affinity using Ba(2+) as the charge carrier have predicted a 10- to 15-fold preference of mibefradil for T channels over L channels. However, T channel IC(50) values are reported to be approximately 1 microM, which is much higher than expected for clinical efficacy because relevant blood levels of this drug are approximately 50 nM. We compared the affinity for mibefradil of the newly cloned T channel isoforms, alpha1G, alpha1H, and alpha1I with an L channel, alpha1C. In 10 mM Ba(2+), mibefradil blocked in the micromolar range and with 12- to 13-fold greater affinity for T channels than for L channels (approximately 1 microM versus 13 microM). When 2 mM Ca(2+) was used as the charge carrier, the drug was more efficacious; the IC(50) for alpha1G shifted to 270 nM and for alpha1H shifted to 140 nM, 4.5- and 9-fold higher affinity than in 10 mM Ba. The data are consistent with the idea that mibefradil competes for its binding site on the channel with the permeant species and that Ba(2+) is a more effective competitor than Ca(2+). Raising temperature to 35 degrees C reduced affinity (IC(50) 792 nM). Reducing channel availability to half increased affinity ( approximately 70 nM). This profile of mibefradil affinity makes these channels good candidates for the physiological target of this antihypertensive agent.

Identification of a T-type Ca(2+) channel isoform in murine atrial myocytes (AT-1 cells)

Calcium channels are important targets for therapeutics, but their molecular diversity complicates characterization of these channels in native heart cells. In this study, we identify a new splice variant of a low-voltage activated, or T-type Ca(2+), channel in murine atrial myocytes. To date, alpha1G and alpha1H are the only 2 T-type Ca(2+) channel isoforms found in cardiovascular tissue. We compared alpha1G and alpha1H channel current heterologously expressed in HEK 293 cells with T-type current from the murine atrial tumor cell, AT-1. AT-1 cell T-type current (I(T)) has the same voltage dependence of activation and inactivation as alpha1G and alpha1H. The cloned T-type channels and AT-1 T-type current share similar kinetics of macroscopic inactivation and deactivation. The kinetics of recovery from inactivation of T-type currents serves as an electrophysiological signature for T-channel isoform. alpha1G and AT-1 I(T) have a similar recovery from inactivation time course that is faster than that for alpha1H. In all cases, T-type current recovers with a biexponential time course, and the relative amplitude of fast and slow time courses explains the slower alpha1H recovery kinetics, rather than differences in the time constants of the individual transitions. Thus, the T-type channels may be an important contributor to automaticity in heart cells, and molecular diversity is reflected in the pathway of recovery from inactivation.

Arachidonic acid modulation of alpha1H, a cloned human T-type calcium channel.

Arachidonic acid (AA) and the products of its metabolism are central mediators of changes in cellular excitability. We show that the recently cloned and expressed T-type or low-voltage-activated Ca channel, alpha1H, is modulated by external AA. AA (10 microM) causes a slow, time-dependent attenuation of alpha1H current. At a holding potential of -80 mV, 10 microM AA reduces peak inward alpha1H current by 15% in 15 min and 70% in 30 min and shifts the steady-state inactivation curve -25 mV. AA inhibition was not affected by applying the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid. The epoxygenase inhibitor octadecynoic acid partially antagonized AA attenuation of alpha1H. The epoxygenase metabolite epoxyeicosatrienoic acid (8,9-EET) mimicked the inhibitory effect of AA on alpha1H peak current. A protein kinase C (PKC)-specific inhibitor (peptide fragment 19-36) only partially antagonized the AA-induced reduction of peak alpha1H current and the shift of the steady-state inactivation curve but had no effect on 8,9-EET-induced attenuation of current. In contrast, PKA has no role in the modulation of alpha1H. These results suggest that AA attenuation and shift of alpha1H may be mediated directly by AA. The heterologous expression of T-type Ca channels allows us to study for the first time properties of this important class of ion channel in isolation. There is a significant overlap of the steady-state activation and inactivation curves, which implies a substantial window current. The selective shift of the steady-state inactivation curve by AA reduces peak Ca current and eliminates the window current. We conclude that AA may partly mediate physiological effects such as vasodilatation via the attenuation of T-type Ca channel current and the elimination of a T-type channel steady window current.

Molecular cloning and functional expression of Ca(v)3.1c, a T-type calcium channel from human brain.

Low voltage-activated T-type calcium channels are encoded by a family of at least three genes, with additional diversity created by alternative splicing. This study describes the cloning of the human brain alpha1G, which is a novel isoform, Ca(v)3.1c. Comparison of this sequence to genomic sequences deposited in the GenBank allowed us to identify the intron/exon boundaries of the human CACNA1G gene. A full-length cDNA was constructed, then used to generate a stably-transfected mammalian cell line. The resulting currents were analyzed for their voltage- and time-dependent properties. These properties identify this gene as encoding a T-type Ca(2+) channel.

Distinct kinetics of cloned T-type Ca2 + channels lead to differential Ca2 + entry and frequency-dependence during mock action potentials.

Voltage-dependent activity around the resting potential is determinant in neuronal physiology and participates in the definition of the firing pattern. Low-voltage-activated T-type Ca2 + channels directly affect the membrane potential and control a number of secondary Ca2 + -dependent permeabilities. We have studied the ability of the cloned T-type channels (alpha1G,H,I) to carry Ca2 + currents in response to mock action potentials. The relationship between the spike duration and the current amplitude is specific for each of the T-type channels, reflecting their individual kinetic properties. Typically the charge transfer increases with spike broadening, but the total Ca2 + entry saturates at different spike durations according to the channel type: 4 ms for alpha1G; 7 ms for alpha1H; and > 10 ms for alpha1I channels. During bursts, currents are inhibited and/or transiently potentiated according to the alpha1 channel type, with larger effects at higher frequency. The inhibition may be induced by voltage-independent transitions toward inactivated states and/or channel inactivation through intermediate closed states. The potentiation is explained by an acceleration in the channel activation kinetics. Relatively fast inactivation and slow recovery limit the ability of alpha1G and alpha1H channels to respond to high frequency stimulation ( > 20 Hz). In contrast, the slow inactivation of alpha1I subunits allows these channels to continue participating in high frequency bursts (100 Hz). The biophysical properties of alpha1G, H and I channels will therefore dramatically modulate the effect of neuronal activities on Ca2 + signalling.

Comparison of the Ca2 + currents induced by expression of three cloned alpha1 subunits, alpha1G, alpha1H and alpha1I, of low-voltage-activated T-type Ca2 + channels.

Expression of rat alpha1G, human alpha1H and rat alpha1I subunits of voltage-activated Ca2 + channels in HEK-293 cells yields robust Ca2 + inward currents with 1.25 mM Ca2 + as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of alpha1G show the fastest activation and inactivation kinetics. The alpha1H and alpha1I currents activate and inactivate up to 1.5- and 5-fold slower, respectively. No differences in the voltage dependence of steady state inactivation are detected. Currents induced by expression of alpha1G and alpha1H deactivate with time constants of up to 6 ms at a test potential of - 80 mV, but currents induced by alpha1I deactivate about three-fold faster. Recovery from short-term inactivation is more than three-fold slower for currents induced by alpha1H and alpha1I in comparison to alpha1G. In contrast to these characteristics, reactivation after long-term inactivation was fastest for currents arising from expression of alpha1I and slowest in cells expressing alpha1H calcium channels. The calcium inward current induced by expression of alpha1I is increased by positive prepulses while currents induced by alpha1H and alpha1G show little ( < 5%) or no facilitation. The data thus provide a characteristic fingerprint of each channel's activity, which may allow correlation of the alpha1G, alpha1H and alpha1I induced currents with their in vivo counterparts.

Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H.

Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents.

The effect of alpha2-delta and other accessory subunits on expression and properties of the calcium channel alpha1G.

1. The effect has been examined of the accessory alpha2-delta and beta subunits on the properties of alpha1G currents expressed in monkey COS-7 cells and Xenopus oocytes. 2. In immunocytochemical experiments, the co-expression of alpha2-delta increased plasma membrane localization of expressed alpha1G and conversely, the heterologous expression of alpha1G increased immunostaining for endogenous alpha2-delta, suggesting an interaction between the two subunits. 3. Heterologous expression of alpha2-delta together with alpha1G in COS-7 cells increased the amplitude of expressed alpha1G currents by about 2-fold. This finding was confirmed in the Xenopus oocyte expression system. The truncated delta construct did not increase alpha1G current amplitude, or increase its plasma membrane expression. This indicates that it is the exofacial alpha2 domain that is involved in the enhancement by alpha2-delta. 4. Beta1b also produced an increase of functional expression of alpha1G, either in the absence or the presence of heterologously expressed alpha2-delta, whereas the other beta subunits had much smaller effects. 5. None of the accessory subunits had any marked influence on the voltage dependence or kinetics of the expressed alpha1G currents. These results therefore suggest that alpha2-delta and beta1b interact with alpha1G to increase trafficking of, or stabilize, functional alpha1G channels expressed at the plasma membrane.

Molecular characterization of two members of the T-type calcium channel family.

In this chapter we review our recent studies on the cloning of two novel cDNAs (alpha 1G and alpha 1H), and present electrophysiological evidence that they encode low voltage-activated, T-type calcium channels (CavT.1 and CavT.2, respectively). The nucleotide sequences of these T channels are very different from high voltage-activated Ca2+ channels, which explains why they were not cloned earlier using homology-based strategies. We used a bioinformatic approach, cloning the first fragment in silico. We then used this fragment to screen human heart and rat brain lambda gt10 libraries, leading to the cloning of two full-length cDNAs derived from distinct genes (CACNA1G and CACNA1H). The deduced amino acid sequences of the T channels (alpha 1G and alpha 1H) are also very different from previously cloned Ca2+ and Na+ channels; however, there are regions of structural similarity. For example, the T channels also contain four repeats, and within each repeat there are six putative membrane-spanning regions and a pore loop. Expression of these cloned channels in either Xenopus oocytes or HEK-293 cells leads to the formation of typical T-type currents. As observed for native T currents, these channels activate at potentials near the resting membrane potential, inactivate rapidly, deactivate slowly, and have a tiny single-channel conductance. The currents generated by alpha 1G and alpha 1H are nearly identical in terms of their voltage dependence and kinetics. We present preliminary evidence that nickel may serve as a valuable tool in discriminating between these subtypes.

Cloning of a novel four repeat protein related to voltage-gated sodium and calcium channels.

Cloning has led to the discovery of more ion channels than predicted by functional studies, yet there remain channels that have not been cloned. We report the cloning of a novel protein that contains the four domain structure found in voltage-gated Ca2+ and Na+ channels. Phylogenetic relationships suggested that the protein might have diverged from an ancestral four repeat channel before the divergence of Ca2+ and Na+ channels. Northern blot analysis showed that mRNA transcripts encoding the protein are expressed predominantly in the brain, moderately in the heart, and weakly in the pancreas. Despite extensive expression attempts, currents from the putative channel were not detected. Based on its sequence, we propose that the novel protein might be a voltage-activated cation channel with unique gating properties.

Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels.

Low voltage-activated (T-type) calcium currents are observed in many central and peripheral neurons and display distinct physiological and functional properties. Using in situ hybridization, we have localized central and peripheral nervous system expression of three transcripts (alpha1G, alpha1H, and alpha1I) of the T-type calcium channel family (CaVT). Each mRNA demonstrated a unique distribution, and expression of the three genes was largely complementary. We found high levels of expression of these transcripts in regions associated with prominent T-type currents, including inferior olivary and thalamic relay neurons (which expressed alpha1G), sensory ganglia, pituitary, and dentate gyrus granule neurons (alpha1H), and thalamic reticular neurons (alpha1I and alpha1H). Other regions of high expression included the Purkinje cell layer of the cerebellum, the bed nucleus of the stria terminalis, the claustrum (alpha1G), the olfactory tubercles (alpha1H and alpha1I), and the subthalamic nucleus (alpha1I and alpha1G). Some neurons expressed high levels of all three genes, including hippocampal pyramidal neurons and olfactory granule cells. Many brain regions showed a predominance of labeling for alpha1G, including the amygdala, cerebral cortex, rostral hypothalamus, brainstem, and spinal cord. Exceptions included the basal ganglia, which showed more prominent labeling for alpha1H and alpha1I, and the olfactory bulb, the hippocampus, and the caudal hypothalamus, which showed more even levels of all three transcripts. Our results are consistent with the hypothesis that differential gene expression underlies pharmacological and physiological heterogeneity observed in neuronal T-type calcium currents, and they provide a molecular basis for the study of T-type channels in particular neurons.

Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family.

Low voltage-activated Ca2+ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, alpha1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels alpha1G and alpha1H and to the high voltage-activated channels formed by alpha1Ebeta3. The alpha1I channels opened after small depolarizations of the membrane similar to alpha1G and alpha1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca2+ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The alpha1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba2+ solutions. Based on its brain distribution and novel gating properties, we suggest that alpha1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

Low-voltage-activated Ca2+ currents are generated by members of the CavT subunit family (alpha1G/H) in rat primary sensory neurons.

Recently, two members of a new family of Ca2+ channel alpha1 subunits, alpha1G (or CavT.1) and alpha1H (or CavT.2), have been cloned and expressed. These alpha1 subunits generate Ba2+ currents similar to the T-type Ca2+ currents present in sensory neurons. Here, we use three methods to investigate whether the T currents of nodosus ganglion neurons are encoded by members of the CavT family. PCR detected the presence of mRNA encoding both alpha1G and alpha1H, as well as a third highly related sequence, alpha1I. In situ hybridizations performed on nodosus ganglia demonstrate a high expression of alpha1H subunit RNAs. Transfection of nodosus ganglion neurons with a generic antisense oligonucleotide against this new alpha1 subunit family selectively suppresses the low-voltage-activated Ca2+ current. The antisense oligonucleotide effect increased with time after transfection and reached a maximum 3 d after treatment, indicating a 2-3 d turnover for the alpha1 proteins. Taken together, these results suggest that the T-type current present in the sensory neurons is mainly attributable to alpha1H channels. In addition, taking advantage of the high specificity of the antisense ON to the cloned channels, we showed that T-type currents greatly slowed the repolarization occurring during an action potential and were responsible for up to 51% of the Ca2+ entry during spikes. Therefore, the antisense strategy clearly demonstrates the role of low-voltage-activated Ca2+ current in affecting the afterpotential properties and influencing the cell excitability. Such tools should be beneficial to further studies investigating physiological roles of T-type Ca2+ currents.

Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family.

Voltage-activated Ca2+ channels exist as multigene families that share common structural features. Different Ca2+ channels are distinguished by their electrophysiology and pharmacology and can be classified as either low or high voltage-activated channels. Six alpha1 subunit genes cloned previously code for high voltage-activated Ca2+ channels; therefore, we have used a database search strategy to identify new Ca2+ channel genes, possibly including low voltage-activated (T-type) channels. A novel expressed sequence-tagged cDNA clone of alpha1G was used to screen a cDNA library, and in the present study, we report the cloning of alpha1H (or CavT.2), a low voltage-activated Ca2+ channel from human heart. Northern blots of human mRNA detected more alpha1H expression in peripheral tissues, such as kidney and heart, than in brain. We mapped the gene, CACNA1H, to human chromosome 16p13.3 and mouse chromosome 17. Expression of alpha1H in HEK-293 cells resulted in Ca2+ channel currents displaying voltage dependence, kinetics, and unitary conductance characteristic of native T-type Ca2+ channels. The alpha1H channel is sensitive to mibefradil, a nondihydropyridine Ca2+ channel blocker, with an IC50 of 1.4 micromol/L, consistent with the reported potency of mibefradil for T-type Ca2+ channels. Together with alpha1G, a rat brain T-type Ca2+ channel also cloned in our laboratory, these genes define a unique family of Ca2+ channels.

Molecular characterization of a neuronal low-voltage-activated T-type calcium channel.

The molecular diversity of voltage-activated calcium channels was established by studies showing that channels could be distinguished by their voltage-dependence, deactivation and single-channel conductance. Low-voltage-activated channels are called 'T' type because their currents are both transient (owing to fast inactivation) and tiny (owing to small conductance). T-type channels are thought to be involved in pacemaker activity, low-threshold calcium spikes, neuronal oscillations and resonance, and rebound burst firing. Here we report the identification of a neuronal T-type channel. Our cloning strategy began with an analysis of Genbank sequences defined as sharing homology with calcium channels. We sequenced an expressed sequence tag (EST), then used it to clone a full-length complementary DNA from rat brain. Northern blot analysis indicated that this gene is expressed predominantly in brain, in particular the amygdala, cerebellum and thalamus. We mapped the human gene to chromosome 17q22, and the mouse gene to chromosome 11. Functional expression of the channel was measured in Xenopus oocytes. Based on the channel's distinctive voltage dependence, slow deactivation kinetics, and 7.5-pS single-channel conductance, we conclude that this channel is a low-voltage-activated T-type calcium channel.

Full reversal of Pb++ block of L-type Ca++ channels requires treatment with heavy metal antidotes.

The mechanisms of Pb++ block and unblock of L-type Ca++ channel currents were measured using ventricular myocytes or the cloned channel. The cloned channel was expressed in either Xenopus laevis oocytes or human embryonic kidney cells (HEK 293, stable transfectants). The threshold for Pb++ block was 1 nM, and the apparent IC50 value was 152 nM in oocytes and 169 nM in HEK 293 cells. Pb++ block was dependent on the composition of the external recording solution but not dependent on the subunit composition of the channel. Pb++ block was voltage dependent, with little block observed at negative test potentials using low concentrations of Pb++. Strong depolarizations (>+100 mV) reversed Pb++ block, allowing measurement of reblock kinetics. Reblock was fast (tau = 11 msec), as measured during a +20-mV test pulse. Simple washout did not completely reverse Pb++ block, especially after exposure to concentrations of >100 nM. Full recovery could only be observed after treatment with heavy metal antidotes such as meso-2,3-dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonic acid and EDTA. These results suggest that Pb++ blocks voltage-gated Ca++ channels by two mechanisms and that full reversal of lead block requires chelator treatment.