I. INTRODUCTION

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Rises in intracellular calcium trigger a variety of processes including muscle contraction, chemotaxis, gene expression, synaptic plasticity, and secretion of hormones and neurotransmitters. Although there are many channels and pumps involved in controlling intracellular Ca²⁺ levels, voltage-gated Ca²⁺ channels play a key role in this process (50). Calcium is not only an important second messenger, but its entry can also depolarize the plasma membrane, and thereby activate other voltage-gated ion channels. This property is especially important for neuronal T-type channels, which can generate low-threshold spikes that lead to burst firing and oscillatory behavior (175, 377). This activity is especially prominent in the thalamus, where it plays an important role in sensory gating, sleep, and arousal (277). The term thalamocortical dysrhythmias has been coined to describe pathological changes in these oscillations, and they have been implicated in a wide range of neurological disorders including absence epilepsy, the tremor associated with Parkinson's disease, tinnitus, neuropsychiatric disorders, and neurogenic pain (186, 242).

The ability of neurons to fire low-threshold Ca²⁺ spikes suggested the existence of low-voltage-activated (LVA) Ca²⁺ channels. Since 1975 it has been recognized that there were at least two distinct types of Ca²⁺ channel (reviewed in Ref. 153). Hagiwara, Ozawa, and Sand, using two-microelectrode voltage-clamp recordings from starfish eggs, found channels that were activated after small depolarizations of the membrane, LVA, and other channels that required larger depolarizations of the membrane, high voltage activated (HVA) (154). LVA currents inactivated faster, more completely, and at more negative membrane potentials than HVA currents. Similarly, LVA and HVA currents were described in the marine pileworm Neanthes (123). Of historical note, the first recordings of mammalian LVA channels were made by Moolenar and Spector in 1978 (290), who used the two-microelectrode voltage-clamp technique on the mouse neuroblastoma cell line N1E-115. However, these authors only observed one type of Ca²⁺ channel, which in 10 mM Ca²⁺ activated at 55 mV and peaked at 20 mV. Subsequent studies have shown that N1E-115 cells provide a convenient system to study the properties of native T-type currents (see sect. III). Using quinidine to block K⁺ currents, Fishman and Spector reported in 1981 (120) on the existence of two types of mammalian Ca²⁺ channel. One type activated at 50 mV, peaked at 20 mV, and inactivated rapidly ( ~15-30 ms). The second type peaked at 0 mV and inactivated slowly ( ~2,000 ms). The existence of LVA currents was firmly established by the work of many groups, including those headed by Kostyuk (115, 420), Carbone and Lux (62), Armstrong (12), Bean (32), Feltz (54), and Tsien (303, 306). Whole cell patch-clamp recordings showed that depolarizations in the 60 to 20 mV range elicited rapidly inactivating currents, while higher depolarizations elicited noninactivating currents. Plots of the current versus test potential (I-V) showed two components, with the LVA component appearing as a hump on the back of a larger HVA component. LVA currents also turned off, or deactivated, slower than HVA currents, producing slow tail currents (62). This property was studied in great detail in GH₃ cells, where slowly deactivating (SD) tail currents were found to activate at lower thresholds than fast deactivating (FD) currents (12). Tsien and colleagues (306) extended the classification of dorsal root ganglion (DRG) channels and proposed that these channels be called T type for transient, L type for long lasting, and N type for neither T nor L type. LVA currents were also found to be resistant to rundown, unlike HVA currents that required cAMP, ATP, and Mg²⁺ in the pipette for stability (115). T- and L-type channels were also described in cardiac myocytes from guinea pig ventricle and dog atrium and could be distinguished by their voltage dependence, kinetics, single-channel currents, pharmacology, and conductance of Ca²⁺ and Ba²⁺ (32, 303). The observation that T-type currents inactivate at lower membrane potentials than L-type currents led to the development of an assay to separate these two components (32). The activity of both channels was recorded from a well-hyperpolarized potential such as 90 mV, then the activity of L-type channels was recorded at a potential where T-type channels are inactivated and L type are not (typically 50 mV), then the L-type currents are subtracted from the T- plus L-type currents to isolate the T-type current. Additional methods, such as tail current analysis, are required to isolate T-type currents in most other tissues due to the presence of HVA channels that also inactivate at 50 mV. Another possible contamination is voltage-gated Na⁺ currents that appear to conduct Ca²⁺ (I_Ca,TTX); however, these can be differentiated by their sensitivity to tetrodotoxin (TTX) (162). Hallmark features of T-type channel electrophysiology are as follows: 1) they begin to open after small depolarizations of the plasma membrane (LVA); 2) their currents during a sustained pulse are transient; 3) they close slowly upon repolarization of the membrane, generating a SD tail current; 4) they have a tiny, and equivalent, single-channel conductance of Ba²⁺ and Ca²⁺; 5) they are relatively insensitive to dihydropyridines; and 6) their steady-state inactivation occurs over a similar voltage range as activation. In fact, T-type channels display a window current, i.e., there is a small range of voltages where T-type channels can open, but do not inactivate completely. This property may be particularly important in controlling intracellular Ca²⁺ levels (45). In conclusion, studies on native channels have provided a toolkit for separating Ca²⁺ channel subtypes, and these tools have proven useful in the characterization of both native and recombinant channels.

T-type channels are expressed throughout the body, including nervous tissue, heart, kidney, smooth muscle, sperm, and many endocrine organs. These channels have been implicated in variety of physiological processes including neuronal firing, hormone secretion, smooth muscle contraction, myoblast fusion, and fertilization. In general, the electrophysiological properties of T-type currents recorded from various cell types are similar, but differences have been noted in how they inactivate and in their pharmacology. This heterogeneity can be explained in part by the existence of three T-type channels that are encoded on separate genes (90, 228, 324).

Voltage-gated Ca²⁺ channels have been classified by their electrophysiological and pharmacological properties, and more recently by their amino acid sequence identity. There are at least 10 genes encoding ₁-subunits of voltage-gated Ca²⁺ channels (Fig. 1). Alignment of their deduced amino acid sequences suggests that gene duplication and divergence of an ancestral Ca²⁺ channel gene gave rise to LVA and HVA subfamilies. Further duplication of the HVA gene gave rise to Ca_v1 and Ca_v2 subfamilies. This duplication must have occurred well over 500 million years ago, since the nematode Caenorhabditis elegans contains one member of each subclass. Eventually the Ca_v1 subfamily evolved into four genes, while both the Ca_v2 and Ca_v3 subfamilies evolved into three. Cloning and expression studies have established that the Ca_v3 family encodes LVA T-type channels (sect. IV), while the Ca_v1 subfamily encodes L-type channels (although expression of Ca_v1.4 has not been reported yet). These channels play a critical role in excitation-contraction coupling in cardiac, skeletal, and smooth muscle. In fact, the Ca_v1.1 gene has evolved beyond a Ca²⁺ channel, and its physiological role in skeletal muscle is as a voltage sensor, coupling membrane depolarization directly to calcium release channels of the sarcoplasmic reticulum (SR) (340). In contrast, cardiac and smooth muscle contraction requires influx of extracellular Ca²⁺ through Ca_v1.2 channels. In heart, this influx activates a larger release of Ca²⁺ from intracellular pools via a mechanism called calcium-induced calcium release (CICR) (37). Ca_v1.2 channels are an important therapeutic target in the control of blood pressure and cardiac rhythm. The physiological roles of Ca_v1.3 channels have been difficult to discern since pharmacological tools have not been described that can separate its activity from Ca_v1.2, and because these channels appear to be coexpressed in a number of tissues. In contrast, the activity of Ca_v2 family members can be selectively blocked by peptide toxins. Splice variation of the Ca_v2.1 gene results in channels that differ in their sensitivity to the spider toxin -Aga-IVA and therefore encode both P- and Q-type channels (55). The Ca_v2.2 gene encodes N-type channels, which are characterized by their irreversible and potent block by the snail toxin -conotoxin-GVIA. Studies with these toxins indicate that Ca_v2.1 and Ca_v2.2 channels mediate the Ca²⁺ influx into presynaptic terminals that trigger neurotransmitter release. In contrast to these channels, it has been difficult to determine which native Ca²⁺ current is carried by Ca_v2.3 channels. Recombinant Ca_v2.3 channels resemble T-type channels in their sensitivity to nickel and their voltage dependence of inactivation, leading to the early suggestion that they might encode T-type channels (376). However, Ca_v2.3 channels require stronger depolarizations for channel opening, i.e., are HVA channels, and they deactivate 10-fold faster than T-type channels (228). Studies with SNX-482, a tarantula toxin that selectively blocks recombinant Ca_v2.3 channels, suggests that these channels mediate R-type currents in some tissues (299).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Evolutionary tree of voltage-gated Ca2+ channels. Tree is based on an alignment of the putative membrane-spanning regions and pore loops of the human channels. Alignment of the full-length sequences yields a similar pattern, although all the branch points are shifted to the left due to inclusion of nonconserved sequences. Low-voltage-activated (LVA) channels appear to have diverged from an ancestral Ca2+ channel before the bifurcation of the high-voltage-activated (HVA) channel family into Cav1 and Cav2 subfamilies. (Adapted from Perez-Reyes E, Cribbs LL, Daud A, Yang J, Lacerda AE, Barclay J, Williamson MP, Fox M, Rees M, and Lee J-H. Molecular characterization of T-type calcium channels. In: Low Voltage-activated T-type Calcium Channels, edited by Tsien RW, Clozel J-P, and Nargeot J. Chester, UK: Adis International, 1998, p. 290-305.)

Electrophysiological studies of recombinant channels show that Ca_v3.1 (formerly _1G) and Ca_v3.2 (_1H) have similar activation and inactivation kinetics, but can be differentiated by their recovery from inactivation and sensitivity to block by nickel (209, 230, 351). The kinetic properties of these ₁-subunits closely resemble native T-type channels. In contrast, recombinant Ca_v1 and Ca_v2 channels require auxiliary subunits for normal gating behavior. This suggests that native T-type channels may be formed by a single ₁-subunit. Ca_v3.3 (_1I) channels also generate LVA currents; however, these currents activate and inactivate much more slowly than T-type channels. Native currents with these properties have only been described in a limited number of studies (19, 99, 177, 316, 398). Although there is no drug that is highly selective (>10-fold) for T-type over other ion channels, block of T-type channels appears to contribute to the therapeutic usefulness of antihypertensives, antiepileptics, anesthetics, and possibly antipsychotics. Therefore, T-type channels provide a novel target for drug development.

	II. ELECTROPHYSIOLOGY OF NATIVE T-TYPE CURRENTS

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Low-threshold calcium spikes (LTS) have been described in slices and in isolated neurons from a variety of brain nuclei such as inferior olive (243, 244), thalamic relay (96, 183), medial pontine reticular formation (146), lateral habenula (437), septum (10), deep cerebellar nuclei (241), CA1-CA3 of the hippocampus (163, 307), association cortex (122), paraventricular (399) and preoptic nuclei of the hypothalamus (385), dorsal raphe (60), globus pallidus (296), and the subthalamic nucleus (40). Typically the spike is crowned by a burst of action potentials (Fig. 2). Addition of TTX to block voltage-gated Na⁺ channels abolishes the fast action potentials, effectively isolating the slowly activating and inactivating LTS. Numerous studies have shown that the LTS is mediated by a Ca²⁺ conductance; it is not observed in Ca²⁺-free external solutions, and it can be blocked by divalent cations such as Co²⁺ and Ni²⁺. Definitive proof that thalamic LTS are mediated by T-type channels was provided by studies on transgenic mice, where knockout of the Ca_v3.1 gene abolished these spikes and burst firing (Fig. 2C) (206).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Low-threshold Ca2+ spikes generate burst firing. A: many neurons can generate two distinct patterns of action potential firing in response to a depolarizing stimulus. Regular, or tonic, firing is elicited when the neuron is depolarized from a resting membrane potential near 55 mV. In contrast, when the membrane potential is below 70 mV, the same depolarizing stimulus triggers a high-frequency burst of action potentials. [From Huguenard JR. Low-voltage-activated (T-type) calcium-channel genes identified. Trends Neurosci 21: 451-452, 1998, with permission from Elsevier Science.] B: a representative example of currents that generate burst firing. Depolarization of the plasma membrane by hyperpolarization-activated current (Ih) leads to activation of T-type currents (IT), and a second phase of depolarization called the low-threshold Ca2+ spike. Riding on top of the low-threshold Ca2+ spike are a burst of Na+ spikes mediated by fast voltage-gated Na+ channels. High-threshold Ca2+ and K+ currents can also be activated by the low-threshold calcium spikes. Ca2+ entry during the burst leads to activation of Ca2+-activated K+ currents, which in combination with voltage-gated K+ channels repolarize the membrane. (From Bal T and McCormick DA. Synchronized oscillations in the inferior olive are controlled by the hyperpolarization-activated cation current Ih. J Neurophysiol 77: 3145-3156, 1997.) C: thalamic neurons of transgenic mice lacking expression of Cav3.1 do not fire bursts. Current-clamp recordings are from neurons held at 60, 70, or 80 mV, then depolarized or hyperpolarized by current injections of varying magnitudes as indicated below the set of traces. When the resting membrane potential is 60 mV, a depolarizing stimulus triggers tonic firing in both wild-type (+/+) and transgenic (/) animals. When the membrane potential (Vm) is lowered to 70 mV, a hyperpolarizing stimulus triggers burst firing in neurons from wild-type, but not transgenic animals. When the resting membrane potential is 80 mV, a depolarizing stimulus only triggers burst firing in neurons from wild-type animals. (From Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, and Shin HS. Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking 1G T-type Ca2+ channels. Neuron 31: 35-45, 2001, with permission from Elsevier Science.)

With a few notable exceptions (7, 191), LTS cannot be triggered by depolarization of the neuron from the resting membrane potential, which is typically between 60 and 65 mV. However, LTS can be observed after a hyperpolarizing pulse is delivered. This process has been called "deinactivation" and is due to recovery of channels from inactivation. Table 1 lists a number of studies where the voltage and time dependence for recovery of these spikes has been examined in detail. LTS began to appear after the neuronal membrane was hyperpolarized below 69 mV, and full-amplitude spikes were observed when the membrane was below 73 mV. The rate of recovery was measured by varying the duration of a hyperpolarizing pulse, then testing for the presence of an LTS upon return to the resting membrane potential. These studies found that the LTS deinactivated relatively quickly, with half of it recovering in ~100 ms and full recovery after 200 ms. Therefore, one physiological role of the LTS is as a pacemaker. Although increases in intracellular concentrations of Ca²⁺ act as a second messenger for a variety of processes, in this case the important property is that influx of the divalent cation leads to direct depolarization of the membrane. The amplitude of this depolarization was typically 25 mV (Table 1), which raised the membrane potential to approximately 40 mV. Since the threshold of many voltage-gated Na⁺ channels is around 55 mV, the LTS can trigger a burst of action potentials. These Na⁺-dependent action potentials are brief (1-2 ms) and of high amplitude (80 mV), and in some thalamic neurons of very high frequency (>400 Hz). High-threshold Ca²⁺ channels will also begin to open at potentials more positive than 40 mV (385). This will lead to more influx of Ca²⁺ and, if present, activation of Ca²⁺-dependent K⁺ channels, which can lead to an afterhyperpolarization (AHP) (40, 244). Low-threshold channels can recover from inactivation during the AHP and could begin to fire again as the membrane potential returns to its resting level. Hyperpolarizations can also activate I_h channels, which allow the influx of both Na⁺ and K⁺, thereby depolarizing the cell and directly triggering another LTS (Fig. 2). In this scenario I_h are the primary pacemaker current (254). Clearly there are a variety of ionic conductances that mediate pacemaker activity, and it is an oversimplification to implicate a single channel as the mediator of a pacemaker current. This is especially true in the heart (61).

Another way that low-threshold channels are involved in generating oscillations includes reciprocal connections with an inhibitory interneuron. Due to its important role in controlling brain activity, the best studied of these circuits involves the GABAergic neurons of the thalamic reticular nucleus that regulate the activity of thalamic relay neurons (377). Interestingly, the firing patterns of thalamic relay neurons vary with the state of consciousness (277, 378). In the awake state and during rapid-eye-movement sleep, they fire in tonic mode: the resting membrane potential is relatively depolarized, the LTS is inactivated, and excitatory inputs faithfully trigger action potentials. In deep sleep, or during thalamocortical dysrhythmias, these neurons fire in an oscillatory mode called burst firing; the resting membrane potential is hyperpolarized, allowing full-amplitude low-threshold spiking (Fig. 2A). Such firing is thought to underlie spike-wave discharges observed during absence seizures (176). Similar patterns of brain activity have been detected in a variety of neurological disorders by magnetoencephalography and have been termed thalamocortical dysrhythmias (242).

B.  Neuronal

1.  Isolated neurons

LVA T-type currents have been characterized using whole cell voltage-clamp recording of neurons isolated from a variety of brain regions. Very large T-type currents (>1 nA) have been recorded from cholinergic neurons of the basal forebrain (6), from relay neurons of the ventrobasal thalamus (222), and from sensory neurons of dorsal root ganglia (436). Prominent T-type currents have been found in neurons from many brain regions (Table 2). I-V relationships are typically measured by holding the cell at 90 mV, and then currents are elicited by depolarizing pulses that increase in 10-mV increments. T-type currents begin to activate when the membrane is depolarized above 60 mV. At threshold potentials, the currents activate relatively slowly, requiring many milliseconds to reach peak, and also inactivate relatively slowly. At higher potentials both activation and inactivation are faster. This produces a stereotypical pattern in the family of current traces where successive records cross each other, reminiscent of voltage-gated Na⁺ channels. In general, HVA channels do not produce this criss-crossing pattern because inactivation rates are much slower than activation rates (334). I-V curves commonly show two components, with the LVA currents peaking at 30 mV while the HVA currents peak around 0 mV. In many neurons, T-type currents can be measured in isolation with test pulses to 30 mV and below. However, at higher potentials both LVA and HVA currents are activated, complicating estimates of the LVA activation curve. Both I-V curves show a peak then decrease at more positive potentials, which is due to a reduction of the electrochemical gradient for calcium ions as the test potential approaches the theoretical reversal potential (~100 mV; see Fig. 7 for example). Therefore, the fraction of channels activated by a test pulse continues to increase beyond the peak of the I-V curve. This explains why estimates of the midpoint of channel activation (V_0.5) derived by normalizing the current at each test potential to the maximum current observed (I/I_max) produces values that are more negative (50 mV) than estimates that take into account changes in driving force (41 mV). To circumvent this problem, activation curves have also been estimated by measuring the amplitude of slowly deactivating tail currents at a constant voltage. Most of these studies used a test pulse of constant duration (isochronal), which assumes that the rates of channel activation and inactivation are voltage independent. However, this is not the case, so this method can underestimate channel activation at negative potentials where channels open slowly. It can also underestimate activation if channels inactivate during the test pulse. This problem can be overcome by varying the duration of the activating pulse, so that the tail current is elicited at the peak of the current (288). So far this method has only been applied to recombinant channels. Another experimental variable that affects the position of the activation curve is the concentration and choice of charge carrier. Millimolar concentrations of positively charged divalent cations can bind to surface charges on both the channel and the membrane, thereby reducing the effective transmembrane voltage gradient (164). An effect called surface charge screening. Increasing external [Ca²⁺] from 2 to 10 mM shifts the apparent gating of T-type channels by ~10 mV (128).

A hallmark of T-type currents is that they are transient; currents reach a peak then decay. In solutions where K⁺ channels are blocked, this decay is due to channel inactivation. The inactivation rate is measured by fitting the current trace with an exponential function and is typically reported with a  in milliseconds. Plots of inactivation rate versus test potential reveal two phases: inactivation is slow at 60, gets faster between 50 and 30, then is constant above 30 mV. Activation rates have a similar voltage dependence, leading to the notion that channels must activate before inactivating, and that the inactivation process is essentially voltage-independent. Although most T-type currents inactivate relatively rapidly (<30 ms), there are also reports of slowly inactivating currents (Table 2). This variability was interpreted as evidence for multiple T-type channel genes (179). This hypothesis was supported by the cloning of T-type channels that differed in this property; Ca_v3.1 and 3.2 display fast inactivation, while Ca_v3.3 displays slowly inactivating currents. Notably, Ca_v3.3 mRNA is expressed in the same brain regions as the slow currents (177, 228, 393). Excluding these cases, the average inactivation  from 22 studies is 20 ms.

The voltage dependence of inactivation is also measured at "steady-state" by holding the membrane at varying potentials (prepulse), then assaying for available T-type channels with a test pulse to 30 mV (h). To approximate steady-state, many studies have used a prepulse duration of 1 s, which is considerably longer than the time required to inactivate open channels. However, increasing the prepulse duration to 10 s results in a 10-mV shift in the apparent h curve (53, 389). Despite the use of different prepulses and concentration of divalent cations, there is good agreement between the 34 studies shown in Table 2, and the average midpoint of the h curve is 77 mV (Table 2). Somewhat surprisingly this value is considerably more negative than the apparent threshold of activation, indicating that channels can inactivate at potentials where they do not appear to open. Similar h values have been obtained with the recombinant Ca_v3 channels (see Table 8), where this closed-state inactivation has been studied in greater detail (127). Although most HVA channels inactivate at more positive potentials, R-type and recombinant Ca_v2.3 channels inactivate over a similar voltage range as T-type channels (334).

The time course of recovery from inactivation is an important property of T-type channels. LTS are often triggered after an inhibitory postsynaptic potential (IPSP), and this is due to fast recovery of T-type channels during the IPSP (deinactivation), followed by their opening as the membrane returns to its resting potential. Recovery is often measured by varying the time between (interpulse) an inactivating pulse and a test pulse. In most studies the interpulse voltage was 90 mV, and similar results are obtained at more negative voltages. In contrast, recovery is slower at potentials where channels can also inactivate. There is considerable variability in the reported values for recovery, ranging from 100 to 3,300 ms (Table 2). Although most studies found that recovery followed a monoexponential time course, others have found evidence for a biexponential process. This discrepancy might be due to differences in the duration of the inactivating pulse, where long pulses allow channels to accumulate in a second inactivated state from which they recover slowly. As observed in sensory neurons (53), recombinant channels recover with a monoexponential time course from short pulses and with a slower, biexponential time course from long pulses, although this property is less pronounced for Ca_v3.3. Differences in recovery kinetics of T-type currents between different neurons can also be due to which Ca_v3 isoform they express (209). Of the three isoforms, Ca_v3.1 channels recover the fastest (~120 ms from short pulses), and this time course is similar to that observed for recovery of both native T-type currents (~300 ms; Table 2) and LTS (~150 ms; Table 1).

Early studies found that LVA channels were more sensitive to block by 50-100 µM nickel than HVA channels (124, 152, 298). Subsequent studies found that many neuronal T-type channels required >10-fold higher concentrations for block, with many studies reporting IC₅₀ values of ~300 µM (Table 2). Studies on recombinant channels provided an explanation for this diversity; only Ca_v3.2 channels are highly nickel sensitive (IC₅₀ ~10 µM), while Ca_v3.1 and Ca_v3.2 are 20-fold less sensitive (230). Recombinant HVA channels also differ in their Ni²⁺ sensitivity, with Ca_v2.3 being relatively sensitive (455). Although the mechanisms of block of recombinant HVA and LVA channels are complex and subject to multiple interpretations, a consistent finding is that block is greatest at potentials near threshold. This voltage dependence would exaggerate block of LVA channels, making nickel appear more selective than it is. In summary, block by 10-50 µM Ni²⁺ can be used to implicate Ca_v3.2 channels, but additional evidence, such as a slowly deactivating tail current, is required to rule out Ca_v2.3 channels. Both these criteria, plus PCR, were recently applied to show the expression of Ca_v3.2 in sympathetic ganglion neurons (231).

The temperature sensitivity of T-type currents has been measured in thalamic neurons (85), DRG neurons (305), N1E-115 neuroblastoma cells (298), and GH₃ cells (343). Heating from 22 to 37°C caused over twofold increases in current amplitudes and accelerated channel activation and inactivation. In contrast, heating did not affect the position of the I-V curve (305). The effects of temperature have been found to be nonlinear, making it difficult to calculate the effect of a 10°C increment (Q₁₀) in temperature (298, 343). In the linear range, Q₁₀ values with an average of 2.5 have been found for effects on amplitude, activation kinetics, and inactivation kinetics.

The pH sensitivity of T-type currents has been studied in CA1 hippocampal neurons (405), ventrobasal thalamic neurons (365), and cardiac myocytes (83, 414). Acidification of the external solution decreased current amplitudes, whereas alkalinization had the opposite effect. In contrast, T-type currents are relatively insensitive to changes in intracellular pH (405). The effect of external pH is in part mediated by changes in the single-channel conductance, which when measured with 110 mM Ca²⁺ increased from 3.5 pS at pH 6 to 10.8 pS at pH 9 (414). Small changes in pH have also been shown to shift the voltage dependence of activation and inactivation; changing pH from 7.3 to 6.9 shifted both of these parameters by +3 mV, while increasing the pH to 7.7 had the opposite effect (365). Larger shifts in the voltage dependence of activation (15 mV) have been observed using larger shifts in pH (from 7.5 to 9.8) (83). The observed pK_a values were ~7, indicating that T-type currents will be affected by modest changes in extracellular pH (365, 414).

LVA currents have been characterized in slices prepared from numerous brain regions (Table 3). The most striking difference between these data and that obtained with isolated neurons is that currents are much larger in slices. This difference has been attributed to loss of channels that were localized on dendrites (97). In fact, T-type channels appear to be preferentially localized to dendrites (77, 135, 195, 199, 328).

T-type currents recorded from slices also appear to activate and inactivate at more negative potentials than observed in isolated neurons. The average threshold in slices was 70 mV, and peak currents were observed at 45 mV (Table 3), while in isolated neurons threshold was 60 and the peak occurred at 30 mV (Table 2). This difference has been attributed to poor voltage clamp of neurons in slices (97). Consistent with this suggestion, the voltage dependence of single T-type channels recorded from dendrites is similar to that observed in isolated neurons (256).

In summary, T-type currents are found in neurons throughout the brain, with particularly large currents found in thalamic, septal, and sensory neurons. Their activation near the resting membrane potential and their fast recovery from inactivation allow them to generate LTS. Their preferential localization in dendrites suggests T-type channels play an important role in synaptic integration.

Cardiac T-type currents have been the focus of many studies due to their possible role as a pacemaker current. These studies have established that T-type current densities are highest in conduction and pacemaker cells, and much reduced or nonexistent in ventricular myocytes. T-type currents are highest near birth and gradually decline but may reappear in pathological conditions. Currents are larger in lower species and have a wider distribution. For example T-type currents are prominent throughout the guinea pig and hamster heart but virtually absent in adult ventricular myocytes of rat, cat, and dog. Although Ca_v3.2 was cloned from a human heart cDNA library, T-type currents have not been detected in human myocytes (39, 235, 313).

Cardiac T-type currents were initially described in dog atrium (32) and guinea pig ventricle (283, 303). These studies showed that cardiac T-type currents resembled those recorded from neurons, but differed from L-type channels in terms of their kinetics (transient), pharmacology (dihydropyridine insensitive), conductance of divalent cations (Ca²⁺ = Ba²⁺), lack of regulation by -adrenergic agonists, and smaller single-channel conductance of Ba²⁺. Subsequent work has established their presence in myocytes isolated from dog Purkinje (367), rabbit sinoatrial node (152), cat atrium and latent pacemaker cells (462), hamster ventricle (362), and rat atrium (446). In general, the currents were small, displaying a peak current density below 3 pA/pF. In contrast, prominent T-type currents (>10 pA/pF) have been recorded from myocytes isolated from shark (271), chick (202), finch (48), and mollusks (452).

Calcium channels were originally classified by their inactivation kinetics: rapidly inactivating or transient channels were called T type, while slowly inactivating, or long-lasting channels were called L type (303, 306). It should be noted that this only holds for currents carried by Ba²⁺, as cardiac L-type currents carried by Ca²⁺ can inactivate at a similar rate as T-type currents. This is due to calcium-induced inactivation of the L-type channel, which appears to be mediated by calmodulin tethered to the carboxy terminus of the channel (111). This inhibition is triggered with every heartbeat as Ca²⁺ entry triggers a larger release of Ca²⁺ from internal stores (37). In contrast, Ca²⁺ currents through T-type channels inactivate a bit slower than Ba²⁺ currents (116, 410), which excludes a similar regulation by calmodulin. Another major difference between these channels is that L-type channels conduct Ba²⁺ threefold better than Ca²⁺, whereas T-type channels conduct both equally well (discussed further in sect. IIJ).

A proposed physiological role for cardiac T-type channels is as a pacemaker current during diastolic depolarization. These studies have been hampered by the lack of a selective blocker, relying heavily on the ability of 40 µM nickel to block T- but not L-type currents (152, 298). Current-clamp studies of spontaneous action potentials showed that nickel slowed the late phase of depolarization, and hence slowed the firing of rabbit sinoatrial nodal cells (SAN). Similar results have been obtained by a number of groups using rabbit SAN (98, 353) or cat latent pacemaker cells (462). Tetramethrin (0.1 µM) was reported to produce selective block as well, and to produce similar effects on pacemaker cycle (152); however, subsequent studies failed to confirm this selectivity (165).

Similarly, a number of studies have found lower sensitivity and selectivity for nickel (Table 4). Two possible explanations for these disparate results are 1) that heart expresses two isoforms of the T-type channel, Ca_v3.1 and Ca_v3.2, and these isoforms differ in their sensitivity to nickel, and 2) that isoform expression is age and species dependent. Cloned Ca_v3.2 channels are blocked at 20-fold lower concentrations than Ca_v3.1 (or Ca_v3.3), displaying an IC₅₀ of ~10 µM (230). There is a strong correlation between nickel sensitivity and Ca_v3.2 expression, suggesting that native Ca_v3.2 channels are as sensitive as the cloned channel (323). This suggests that nickel (<100 µM) can be used to implicate Ca_v3.2 channel expression. Expression in atrium appears to be species specific: Ca_v3.1 appears to predominate in rat and mice (91, 236), while in guinea pigs it is Ca_v3.2 (323). In fact, careful measurement of the nickel dose response in guinea pig atrium revealed a biphasic response, with 80% of the channels being blocked with an apparent IC₅₀ of 23 µM, while 20% of the channels were inhibited with an IC₅₀ of 1,350 µM. Rabbit and cat SAN cells appear to predominantly express Ca_v3.2 channels. In contrast, in situ hybridization of mouse heart suggests that Ca_v3.1 channels predominate, although both isoforms were readily detected and both were enriched in SAN relative to atrium (49). It should be noted that distinct in situ probes might differ in their ability to detect their cognate sequence, making such comparisons difficult. PCR amplification of mouse heart detected the expression of a splice variant of Ca_v3.1 that differs in the III-IV loop (91).

Expression of T-type currents in developing heart has also been studied. T-type currents are readily detectable in neonatal hearts, increase slightly to a peak between postnatal days 4 and 8, then decline slowly to a steady-state (236, 246). In other studies T-type currents were only detected in neonatal rats, disappearing by postnatal day 21 (236). In contrast to L type, the biophysical properties of T-type currents are unchanged during neonatal development. Changes in nickel sensitivity have not been investigated but may be informative. No difference in T-type current density was detected in SAN myocytes from newborn (3-10 days old) and adult (41-48 days old) rabbits (329). Expression of Ca_v3.2 mRNA is higher in fetal human heart than adult, whereas Ca_v1.2 shows the opposite pattern (331).

Cardiac hypertrophy appears to trigger a return to the neonatal pattern of gene expression (388), leading to reexpression of T-type channels in rat and cat ventricular myocytes (173, 268, 308). RNase protection assays indicate that Ca_v3.1 transcripts are upregulated in a rat model where hypertrophy is induced by infarction (173). Although levels of Ca_v3.2 transcripts were not examined, the sensitivity of reexpressed currents to nickel block suggests a contribution of Ca_v3.2 channels as well (173, 268). T-type currents are also reexpressed in dedifferentiated rat ventricular myocytes (114). T-type currents have been found to be increased twofold in cardiomyopathic Syrian hamsters relative to other hamster strains (46, 362). Studies using 8-mo-old animals found that the voltage dependence of activation and inactivation was shifted to more negative potentials in ventricular myocytes from cardiomyopathic animals (362). In contrast, studies using 1-day-old hamsters found that only inactivation was shifted (46). The relevance of these observations to human pathologies is unclear as T-type currents have not been detected in myocytes isolated from normal atrium (235) or diseased ventricle (39, 337).

Calcium influx through voltage-gated channels activates intracellular Ca²⁺ release channels, leading to larger elevations in intracellular Ca²⁺ and hence muscle contraction. The role of L-type channels in this process is well established. T-type channels are also capable of triggering this CICR, albeit much more weakly in both cardiac (370, 461) and skeletal muscle (133). T-type current-induced contraction developed more slowly, suggesting that these channels are not localized near SR stores as are L-type channels. This observation coupled with the lower expression of T-type channels in working myocytes indicates that they do not play a major role in excitation-contraction coupling. Consistent with this notion, mibefradil has little effect on cardiac inotropy at doses that lower blood pressure (356). In contrast, T-type channels may be localized near SR in atrial pacemaker cells of the cat (180). These authors suggested a novel pacemaking mechanism where Ca²⁺ influx through the T-type channel triggers subsarcolemmal Ca²⁺ sparks, which in turn activate Na⁺/Ca²⁺ exchange currents that depolarize the membrane to threshold.

A role for T-type channels in triggering atrial natriuretic peptide (ANP) secretion has been inferred from the effects of mibefradil (236, 434). Evidence that mibefradil acted on T-type channels was provided by the observation that L-type blockers were much less potent blockers, or stimulated ANP secretion.

Relatively little is known about the physiological roles of T-type channels in kidney. T-type currents have only been recorded from smooth muscle cells (SMC) isolated from rat interlobular and arcuate arteries (143). Heterogeneity in Ca²⁺ currents was observed, with approximately one-third of the freshly isolated myocytes displaying only T-type currents ("T-rich"), one-third only L type, and the rest a mixture. Currents in T-rich cells were some of the largest ever recorded for cardiovascular smooth muscle (156 pA in 2.5 mM Ca²⁺; Table 5), which allowed their recording in physiological Ca²⁺ solutions. Consistent with their assignment as T-type channels, currents activated and inactivated at voltages 20 mV more negative than observed for L-type currents. In contrast to most T-type currents, these SMC currents activated (I-V peak 10 mV) and inactivated (h = 50 mV) at voltages that were slightly more positive, which might be characterized as mid-voltage activated. They also inactivated about twofold slower during a pulse (_inact = 46 ms at 10 mV). Atypical currents with similar properties have been reported for SMC isolated from rat portal vein (315) and from the terminal branches of guinea pig mesenteric arteries (291).

Quite surprisingly, the human tissue that expresses the most mRNA for Ca_v3.2 is the kidney (90, 438). In contrast, both Ca_v3.1 and Ca_v3.3 are predominantly expressed in brain (228, 288, 289, 324). Skøtt and co-workers (11, 156) have studied the distribution Ca_v3.1 and 3.2 in kidney and concluded that Ca_v3.1 was in the tubules, while Ca_v3.2 was in renal smooth muscle (11, 156). RT-PCR indicated that mRNAs for Ca_v3.2, and to a lesser extent Ca_v3.1, were expressed in rat (but not rabbit) glomerular afferent and efferent vessels, and vasa recta. The same study reported that K⁺-induced contraction of perfused rabbit afferent arterioles could be blocked by low concentrations of mibefradil (IC₅₀ ~10 nM) that should be selective for T-type channels. Nickel could also block this response; however, the concentrations required (1 mM) would be expected to block almost all Ca²⁺ channels. Similar results were obtained with rat isolated perfused hydronephrotic kidneys: either 1 µM mibefradil or 100 µM nickel (a relatively selective dose) dilated afferent and efferent arterioles constricted with ANG II (314). Selectivity of mibefradil for T-type channels was demonstrated by coadministration with the L-type blocker nifedipine. In these experiments 1 µM nifedipine caused a modest dilation of the efferent arteriole but did not prevent the more pronounced dilation induced by mibefradil.

Ca_v3.1 was detected in dot blots of human fetal, but not adult, kidney (288). RNase protection assays of rat kidney regions indicated that Ca_v3.1 was predominantly expressed in the inner medulla (11). RT-PCR of microdissected nephron segments indicated that Ca_v3.1 was expressed in distal convoluted tubules, connecting tubules, and collecting ducts. This study also examined the distribution of Ca_v3.1 protein using a polyclonal antibody raised against the first 22 amino acids of the deduced Ca_v3.1 sequence (11). Immunoreactivity was detected in the apical domains of the distal convoluted tubules and in principal cells of connecting tubules and inner medullary collecting ducts. Preincubation of the antibody with peptide blocked the signal; however, Western blots were not presented to demonstrate the selectivity of the antibody. These results suggested that T-type channels, along with endothelial calcium channels (ECaC), might be involved in Ca²⁺ reabsorption.

Calcium channel blockers are widely used in the treatment of hypertension. It is generally accepted that they work by blocking L-type channels in vascular smooth muscle, leading to vasodilation. Recent studies suggest that part of their effect may be mediated by changes in renal hemodynamics (172). In intact dogs, both nifedipine and mibefradil increased renal blood flow, whereas only nifedipine affected glomerular filtration rate. Mibefradil has also been reported to decrease renin secretion in rats with renal artery clips, but had no effect on renin secretion from isolated juxtaglomerular cells (423). In conclusion, these studies suggest that T-type channels on afferent and efferent glomerular arterioles may be an important therapeutic target (89). A possible advantage to a T-selective antihypertensive drug is that it may also prevent glomerular damage (192).

In addition to L-type currents, SMCs isolated from arteries, veins, and organs have also been reported to have LVA currents. With the exception of the kidney results noted above, cardiovascular SMCs have tiny LVA currents. Therefore, most studies have had to use very high concentrations of charge carrier, which shifts gating to positive potentials, and currents have only been characterized in terms of their I-V relations and sensitivity to holding potential. These characteristics are not sufficient to establish an LVA current as T type, since HVA currents actually represent a spectrum of mid- to high-voltage-activated currents. HVA currents also inactivate over a wide range of potentials, with some R-type currents inactivating over the same voltage as T-type channels. These concerns take on additional weight with the finding that some vascular SMCs express non-L-type HVA currents (291) such as P-type currents (155). Native P-type currents can be mid-voltage activated (33). In addition, a nickel- and mibefradil-sensitive LVA current has been described in colonic myocytes that gates similar to T-type channels but has different permeability properties (214). LVA channels can be classified as T type by their slow tail currents, which surprisingly have not been reported, and by their single-channel properties, which have been reported (34, 130, 450). Because T-type channels inactivate at the resting membrane potentials of most SMCs (75 to 60 mV, reviewed in Ref. 168), it has been hard to discern their physiological role. Perhaps they contribute to basal intracellular Ca²⁺ concentrations via their window current, or after transient hyperpolarizations induced by spontaneous transient outward currents (STOCs).

LVA currents have been found in the following arterial SMCs: coronary (130, 281, 332), aortic (3), mesenteric (150, 311, 372), rabbit ear (34), rat tail (327, 428), and middle cerebral arterioles (167). RT-PCR detected the expression of both Ca_v3.1 and Ca_v3.2 in rat mesenteric arterioles (150). Single-channel studies have established the presence of 7.5-pS T-type channels in freshly isolated guinea pig coronary SMCs (130). This study also reported that whole cell LVA currents could only be observed in half of the cells. In contrast, LVA currents were never detected in freshly isolated coronary SMCs from rabbits (269) or humans (332). LVA currents were found if the cells were cultured, and these currents appear to be T type based on their voltage dependence and 1:1 conductance of Ca²⁺ and Ba²⁺ (332). Similar results have been obtained with aortic SMCs; T-type currents are expressed in proliferating cultures, but neither in confluent cultures (3, 339) nor in freshly isolated cells (220). In fact, T-type currents were only detected in cells that were in G₀ or G₁ phases (220), leading to the hypothesis that they might play a role in proliferating SMCs (338). Consistent with this notion is the finding that mibefradil could reduce neointima formation following balloon injury to rat carotid arteries (355). However, it should be noted that T-type currents were not observed in SMCs prepared from injured rat aorta, although a transient downregulation of L-type channels was documented (333).

LVA currents have also been detected in SMCs isolated from veins, such as saphenous (450), portal (246), and azygous (282). T-type single-channel currents were recorded in saphenous vein SMCs (450). Isradipine (PN 200-110) was found to block T-type currents of rat portal vein with an IC₅₀ of 0.5 µM, while L-type currents were blocked at 1 nM (246). The sensitivity of these T-type currents was greater than observed in other preparations (see sect. VA), suggesting that Ca_v3 isoforms may differ in their pharmacology. Current-clamp recordings revealed that portal vein SMCs could fire action potentials that resembled those observed with cardiac myocytes (246). Isradipine (10 nM) inhibited the plateau phase with no effect on the rising phase, suggesting that T-type channels might play a pacemaker role in these cells.

LVA currents have been described in SMCs from bronchi (185, 448), ileum (373), colon (445), bladder (382), and uterus (453). The results with pregnant human uterine SMCs are notable because the T-type currents (3 pA/pF in 1.8 mM Ca²⁺) were larger than the L-type currents (453). These currents activated and inactivated (h = 70 mV) at potentials typical of most T-type currents (Table 5). Current-clamp recordings indicated that a depolarizing pulse could trigger an action potential; however, the threshold (20 mV) was higher than observed with neuronal LTS. Sizable (200 pA) T-type currents were also described for porcine bronchial smooth muscle, being detected in 30% of the cells, but absent from tracheal SMCs (448). LVA currents (1.4 pA/pF) were found to disappear when bladder SMCs were cultured (382). Concomitant with this loss was a shift in the threshold for action potential generation from 25 to 15 mV, suggestive of a role for T-type channels.

Newborn rodent skeletal muscle was also used in early studies to establish the existence of T-type channels as separate from L-type channels (30, 81). Developmental studies of mice and rats have shown that the T-type current is only found in embryonic and newborn muscle and disappears by 3 wk of age (29, 38, 142). Concomitantly, the slow L-type current increases. Studies on the muscular dysgenesis (mdg) mouse clearly established that the T- and L-type channels were encoded on separate genes (30, 67, 212), and it is the L-type channel (Ca_v1.1) that plays a critical role in excitation-contraction coupling (395). Recent studies have shown that the T-type current is important for myoblast fusion (45). In particular, it was shown that T-type channels could generate sufficient window currents to alter intracellular Ca²⁺ concentrations, and trigger fusion. This hypothesis was further supported by pharmacological experiments using low concentrations of Ni²⁺ and amiloride, and by antisense oligonucleotides directed against Ca_v3.2 (45). The selective expression of Ca_v3.2 in skeletal muscle fibers has also been demonstrated using single-cell PCR (38). The electrophysiological properties of these currents (Table 4) and Ni²⁺ sensitivity closely resemble recombinant Ca_v3.2 currents, although differences have been noted in kinetics of activation, inactivation, and recovery from inactivation (38). Early studies also noted kinetic differences as a function of development (142), suggesting that these differences are not due to rapid events such as phosphorylation, but rather slower events such as gene transcription of auxiliary subunits (see sect. IVE).

Calcium channels appear to play a role in mediating the egg-induced acrosome reaction that precedes fusion (for review, see Ref. 94). T-type channels have been implicated in this process, although other Ca²⁺ conductances may be involved (134, 435). One reason for this uncertainty is that it is virtually impossible to patch clamp mature sperm (335). Therefore, electrophysiological studies have used spermatogenic cells, which are primary spermatocytes in the pachytene stage. The only voltage-dependent Ca²⁺ channels in these cells are T type, and their biophysical (350) and pharmacological properties (15) have been well characterized. PCR results indicate that Ca_v3.2 is the predominant isoform expressed in human testis (182, 375). Consistent with this result is the high sensitivity (IC₅₀ = 34 µM) with which nickel blocks the spermatocytic T-type current (15), although it should be noted that Sertoli cells also express nickel-sensitive T-type currents (225). Similar concentrations of nickel also block the acrosome reaction (121, 375). Similarly, the following compounds have been found to block spermatocyte T-type channels and the acrosome reaction at similar concentrations: isradipine (PN 200-110), nifedipine, pimozide, amiloride, mibefradil, W-7, and trifluoperazine (13, 15, 121, 247, 350, 375). Spermatocytic T-type channels appear to be regulated by tyrosine and calmodulin-dependent protein kinases (14, 247).

H.  Endocrine Tissues

1.  Adrenal

T-type channels have been implicated in the secretion of aldosterone (76) and cortisol (109). Calcium currents, and their regulation by secretagogues, have been extensively studied in cells isolated from bovine adrenal zona glomerulosa and fasciculata. Glomerulosa cells express a mixture of T- and L-type channels (82), whereas fasciculata cells predominantly express T-type channels (22, 287). Although these cells can be considered nonexcitable due to their lack of Na⁺ channels, they are still capable of generating Ca²⁺-dependent action potentials (22). In contrast, adrenal chromaffin cells express HVA Ca²⁺ and Na⁺ currents but no LVA currents (16).

Serum K⁺ is regulated by secretion of aldosterone from adrenal glomerulosa cells. Glomerulosa cells are excellent K⁺ sensors and depolarize after small increases in serum K⁺, which in turn leads to the activation of T-type channels, Ca²⁺ influx, and stimulation of aldosterone synthesis and release. For example, increasing K⁺ from 2 to 5 mM causes the resting membrane potential of isolated cells to depolarize from 97 to 78 mV (76). Aldosterone secretion is also stimulated by ANG II, an effect mediated by stimulation of T-type channel activity (discussed in sect. IIIB).

Cortisol secretion from adrenal zona fasciculata cells is regulated by ACTH, which depolarizes these cells by inhibiting a K⁺ conductance (286). Depolarization would activate T-type channels, leading to a rise in intracellular Ca²⁺, and ultimately cortisol synthesis and secretion. Cortisol secretion can be inhibited by a variety of blockers including mibefradil, and this block occurs over the same concentration range as their block of the T-type channels (109, 141, 345). ACTH may also regulate the expression of T-type channels (22).

Adrenal T-type channels are encoded by Ca_v3.2. In situ hybridization of rat and bovine glands detected high levels of Ca_v3.2 expression in the cortex, with only trace amounts of Ca_v3.1 and Ca_v3.3 (358). The biophysical properties and sensitivity to Ni²⁺ block of both glomerulosa and fasciculata cells are also consistent with their being encoded by Ca_v3.2 (287, 358).

LVA Ca²⁺ currents have been recorded from cells of the anterior and intermediate lobes of the pituitary gland including (84, 392, 439) lactotropes (240), corticotropes (261), gonadotropes (379), and thyrotropes (204). These cells generate spontaneous action potentials and Ca²⁺ spikes, leading to secretion of hormone (see Ref. 406 and references therein). Hormonal regulation of these LVA currents supports the idea that these currents are involved in secretion (see sect. III). The voltage- and time-dependent properties of these currents are similar, but not identical to other native T-type currents (Tables 2 and 4). Notably the peak of the I-V curve is slightly depolarized. In addition, one study reported that the LVA current was fivefold larger in Ba²⁺ than Ca²⁺ (379), a property typically ascribed to HVA currents. As suggested previously, not all LVA currents are carried by T-type channels, and other criteria must be applied (19, 33, 376). One such discriminating property is their tail deactivation kinetics, and slowly deactivating T-type currents are clearly present in pituitary cells (84). Similarly, these channels can be distinguished at the single-channel level by their conductance for Ba²⁺, and again, pituitary T-type currents have been clearly identified (203). In situ hybridization studies suggest all three Ca_v3 isoforms are expressed, although Ca_v3.2 was the predominant isoform detected (393). Consistent with this result, T-type currents were reported to be nickel sensitive, with 80% of the current blocked by 100 µM NiCl₂ (240). Currents recorded from GH₃ cells are not nickel sensitive (IC₅₀ = 777 µM), suggesting that this tumor-derived cell line most likely expresses Ca_v3.1 channels (160). In conclusion, T-type channels appear to play a pacemaker role in anterior pituitary, although they may also be involved in stimulus-secretion coupling (258).

T-type channels appear to play a similar pacemaker role in insulin secretion from pancreatic -cells of the islets of Langerhans. Increases in plasma glucose and its metabolism in -cells leads to increases in cellular ATP and inhibition of K_ATP channels (300). Closure of K_ATP channels initiates the pacemaker cycle by depolarizing the -cell membrane to about 55 mV. Riding on top of this slow plateau depolarization are rapid calcium-dependent spikes (reviewed in Ref. 352). Insulin secretion can be blocked by a wide variety of agents, indicating that these spikes activate L-, P-, and R-type currents (238, 258, 418).

T-type currents in pancreatic -cells have been characterized at the whole cell (Table 4; Refs. 25, 320) and single-channel level (17, 348). Expression is species dependent, with little or no expression in rodents, but readily detectable in humans (25, 426). Therefore, it is notable that LVA currents were found in -cells from diabetes-prone NOD mice (426). The expression of LVA currents in mouse cells has been reported to be stimulated by a 6-h treatment with cytokines (25 U/ml of interleukin-1 plus 300 U/ml of interferon-; Ref. 427). However, the I-V curve for the cytokine-induced current peaked between +10 and +20, while under similar recording conditions this group found that bona fide, slowly deactivating, T-type currents peaked between 20 and 10 mV (426). Human -cells express both LVA and HVA currents, and in 20% of the cells tested, the LVA current was dominant (25). Voltage-clamp recordings indicated that 100 µM Ni²⁺ could block the LVA current, while current-clamp recordings indicated that it slowed action potential frequency. Similar studies using the rat insulinoma cell line INS-1 found that low concentrations of nickel could completely block T-type currents (IC₅₀ = 30 µM) and partially block insulin secretion (42). Nickel-sensitive T-type currents (IC₅₀ = 3 µM) have also been recorded from the mouse insulinoma cell line NIT-1 (426). Li and co-workers (463) also cloned a splice variant of Ca_v3.1 from an INS-1 cDNA library. The high sensitivity to nickel block suggests that Ca_v3.2 channels are also expressed, and this conclusion is supported by Northern analysis (438).

T-type currents have been reported in a number of cell lines such as 3T3 fibroblasts (73) and many lines of tumor origin, such as neoplastic B lymphocytes (128), the related mouse neuroblastoma-derived lines N1E-115 (298, 368), NG108-15 (196, 334), 140-3 (144), N18 (397), and ND7-23 (213); human neuroblastoma lines IMR-32 (65) and SK-N-MC (18); rat pituitary lines GH₃ (160, 270); human TT cells (also called h-MTC) and rat 6-23 (clone 6) cells from medullary thyroid tumors (43, 44); human Y79 retinoblastoma cells (24); AT-1 from mouse atrium (351); rat smooth muscle lines A7r5 and A10 (272); and the pancreatic insulinoma lines INS-1 (rat; Ref. 42), HIT-T15 (hamster; Ref. 259), and NIT-1 (mouse; Ref. 426). Many of these cell lines were reported to express almost exclusively T-type currents, allowing characterization of these channels in the absence of contaminating HVA currents (Table 6). The study of T-type currents in cell lines has advanced our understanding of gating, permeation, and pharmacology (73, 160, 368). For example, Chen and Hess (73) developed models of gating using recordings from 3T3 fibroblasts. They also showed that the T-type currents of NG108-15 cells had different kinetics, leading them to predict the existence of multiple channel isoforms. A similar conclusion was reached from studies on TT cells, which also recovered from inactivation much more slowly than observed previously (44). Differences in the pharmacology of T-type channels also suggested the existence of distinct channel isoforms (160). The cloning of multiple Ca_v3 isoforms that differ in their kinetics, pharmacology, and recovery from inactivation supports this hypothesis (209, 228, 404).

Single-channel studies provided conclusive evidence that T-type channels were distinct from HVA Ca²⁺ channels (63, 303, 306). T-type channels were distinguished by their smaller currents, insensitivity to dihydropyridines, and voltage dependence of activation and inactivation. In addition to providing criteria for resolving T-type currents, single-channel studies have provided insights into both hormonal regulation of activity and gating.

With isotonic Ba²⁺ as the charge carrier (110 mM), the single-channel current at a test potential of 0 mV is 0.3 pA for T-type channels (Table 7) and 1.5 pA for L-type channels. Single-channel conductance is defined as the slope of the line relating single-channel amplitudes versus test potential and is commonly reported in picoseimens (pS). When recorded in Ba²⁺, the conductance of T-type channels is 7-8 pS (Table 7), which is smaller than that observed for either N-type (13-15 pS) or brain L-type channels (25-28 pS) (118, 125, 417). Somewhat surprisingly, all three families have a similar Ca²⁺ conductance. For example, the L-type channel conductance drops threefold in Ca²⁺ (149, 367). Recombinant Ca_v2.1 and Ca_v2.2 channels also preferentially conduct Ba²⁺ (>2-fold), whereas Ca_v2.3 conducts both ions equally well (56). In contrast, native T-type channels conduct Ca²⁺ and Ba²⁺ (and Sr²⁺) equally well (64, 104, 367, 368). The conductance plots differ in their relative position, with T-type channels gating at more negative ranges, and extrapolating to a reversal potential of approximately +40 mV, while HVA currents appear to reverse at +60 mV (34, 52, 118, 124, 194, 203, 213, 367, 417).

Estimates of permeability based on reversal potentials yield the opposite rank order, with Ca²⁺ being more permeable than Ba²⁺ (128). Similar results have been obtained using recombinant T-type (363) and native L-type channels (161) and suggest that Ca²⁺ binds with high affinity in the pore and permeates when displaced by a second Ca²⁺. A very interesting property of all voltage-gated Ca²⁺ channels is that they become highly permeable to Na⁺ in the absence of Ca²⁺. Apparently Na⁺ are not capable of displacing the bound Ca²⁺, and hence do not permeate. This block is unidirectional, since monovalent cations can displace Ca²⁺ bound in the pore when they approach from the intracellular side of the channel (255, 363). The selectivity sequence for monovalent cations through T-type channels is Li⁺ Na⁺ > K⁺  Rb⁺ > Cs⁺ (128, 255). In fact, T-type channels begin to pass outward K⁺ currents at physiological concentrations of Ca²⁺ and voltage (+28 mV; Ref. 128). HVA channels can also pass outward currents, but this requires depolarization of the membrane beyond +70 mV (161). Our understanding of Ca²⁺ channel permeation is far from complete, and subject to multiple