Antigen retrieval pre-treatment causes a different expression pattern of Cav3.2 in rat and mouse spinal dorsal horn.

Cav3 channels consist of three isoforms, Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I), which produce low-threshold spikes that trigger burst firings in nociceptive neurons of the spinal dorsal horn (SDH) and dorsal root ganglion (DRG). Although Cav3.2 plays a crucial role in pathological pain, its distribution in SDH still remains controversial. One study showed that Cav3.2 is ubiquitously expressed in neurons, but another study implied that Cav3.2 is expressed restricted to astrocytes. To unravel these discrepancies, we used methods of immunohistochemistry either with or without antigen retrieval (AR) pre-treatment to detect Cav3 in SDH and DRG from both rats and mice. Moreover, Cav3.2 mRNA was detected in mice SDH using in situ hybridization. We found that the expression pattern of Cav3.2 but not Cav3.1 and Cav3.3 in SDH were largely different with or without AR pre-treatment, which showed a neuron-like and an astrocyte-like appearance, respectively. Double staining further demonstrated that Cav3.2 was mainly co-stained with the neuronal marker NeuN in the presence of AR but was with glial fibrillary acidic protein (GFAP, marker for astrocytes) in the absence of AR pre-treatment. Importantly, Cav3.2 mRNA was mainly co-localized with Cav3.2 but not GFAP. Together, our findings indicate that AR pre-treatment or not impacts the expression pattern of Cav3.2, which may make a significant contribution to the future study of Cav3.2 in SDH.

Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue.

Four-domain voltage-gated Ca2+ (Cav) channels play fundamental roles in the nervous system, but little is known about when or how their unique properties and cellular roles evolved. Of the three types of metazoan Cav channels, Cav1 (L-type), Cav2 (P/Q-, N- and R-type) and Cav3 (T-type), Cav3 channels are optimized for regulating cellular excitability because of their fast kinetics and low activation voltages. These same properties permit Cav3 channels to drive low-threshold exocytosis in select neurons and neurosecretory cells. Here, we characterize the single T-type calcium channel from Trichoplax adhaerens (TCav3), an early diverging animal that lacks muscle, neurons, and synapses. Co-immunolocalization using antibodies against TCav3 and neurosecretory cell marker complexin labeled gland cells, which are hypothesized to play roles in paracrine signaling. Cloning and in vitro expression of TCav3 reveals that, despite roughly 600 million years of divergence from other T-type channels, it bears the defining structural and biophysical features of the Cav3 family. We also characterize the channel's cation permeation properties and find that its pore is less selective for Ca2+ over Na+ compared with the human homologue Cav3.1, yet it exhibits a similar potent block of inward Na+ current by low external Ca2+ concentrations (i.e., the Ca2+ block effect). A comparison of the permeability features of TCav3 with other cloned channels suggests that Ca2+ block is a locus of evolutionary change in T-type channel cation permeation properties and that mammalian channels distinguish themselves from invertebrate ones by bearing both stronger Ca2+ block and higher Ca2+ selectivity. TCav3 is the most divergent metazoan T-type calcium channel and thus provides an evolutionary perspective on Cav3 channel structure-function properties, ion selectivity, and cellular physiology.

Dehydroepiandrosterone (DHEA) inhibits voltage-gated T-type calcium channels.

BACKGROUND AND PURPOSE: Dehydroepiandrosterone (DHEA) and its sulfated form, DHEAS, are the most abundant steroid hormones in the mammalian blood flow. DHEA may have beneficial effects in various pathophysiological conditions such as cardiovascular diseases or deterioration of the sense of well-being. However to date, the cellular mechanism underlying DHEA action remains elusive and may involve ion channel modulation. In this study, we have characterized the effect of DHEA on T-type voltage-activated calcium channels (T-channels), which are involved in several cardiovascular and neuronal diseases. KEY RESULTS: Using the whole-cell patch-clamp technique, we demonstrate that DHEA inhibits the three recombinant T-channels (Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3) expressed in NG108-15 cell line, as well as native T-channels in pulmonary artery smooth muscle cells. This effect of DHEA is both concentration (IC(50) between 2 and 7µM) and voltage-dependent and results in a significant shift of the steady-state inactivation curves toward hyperpolarized potentials. Consequently, DHEA reduces window T-current and inhibits membrane potential oscillations induced by Ca(V)3 channels. DHEA inhibition is not dependent on the activation of nuclear androgen or estrogen receptors and implicates a PTX-sensitive Gi protein pathway. Functionally, DHEA and the T-type inhibitor NNC 55-0396 inhibited KCl-induced contraction of pulmonary artery rings and their effect was not cumulative. CONCLUSIONS: Altogether, the present data demonstrate that DHEA inhibits T-channels by a Gi protein dependent pathway. DHEA-induced alteration in T-channel activity could thus account for its therapeutic action and/or physiological effects.

Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis.

Here we describe features of the first non-mammalian T-type calcium channel (LCa(v)3) expressed in vitro. This molluscan channel possesses combined biophysical properties that are reminiscent of all mammalian T-type channels. It exhibits T-type features such as "transient" kinetics, but the "tiny" label, usually associated with Ba(2+) conductance, is hard to reconcile with the "bigness" of this channel in many respects. LCa(v)3 is 25% larger than any voltage-gated ion channel expressed to date. It codes for a massive, 322-kDa protein that conducts large macroscopic currents in vitro. LCa(v)3 is also the most abundant Ca(2+) channel transcript in the snail nervous system. A window current at typical resting potentials appears to be at least as large as that reported for mammalian channels. This distant gene provides a unique perspective to analyze the structural, functional, drug binding, and evolutionary aspects of T-type channels.

T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids.

Lipoamino acids are anandamide-related endogenous molecules that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacological targets underlying their physiological effects. Various lipoamino acids, including N-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC(50) approximately 200 nm for N-arachidonoyl 3-OH-gamma-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addition, we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.

Five different profiles of dihydropyridines in blocking T-type Ca(2+) channel subtypes (Ca(v)3.1 (alpha(1G)), Ca(v)3.2 (alpha(1H)), and Ca(v)3.3 (alpha(1I))) expressed in Xenopus oocytes.

1,4-dihydropyridine (DHP) Ca(2+) antagonists have recently been shown to block T-type Ca(2+) channels, which may render favorable actions on cardiovascular systems. However, this evaluation remains to be done systematically for each T-type Ca(2+) channel subtype except for the Ca(v)3.1 (alpha(1G)) subtype. To address this issue at the molecular level, blocking effects of 14 kinds of DHPs (amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine), which are clinically used for treatments of hypertension, on 3 subtypes of T-type Ca(2+) channels [Ca(v)3.2 (alpha(1H)), Ca(v)3.3 (alpha(1I)), and Ca(v)3.1 (alpha(1G))] were investigated in the Xenopus oocyte expression system using the two-microelectrode voltage-clamp technique. These 3 kinds (alpha(1H), alpha(1I) and alpha(1G)) of T-type channels were blocked by amlodipine, manidipine and nicardipine. On the other hand, azelnidipine, barnidipine, benidipine and efonidipine significantly blocked alpha(1H) and alpha(1G), but not alpha(1I) channels, while nilvadipine and nimodipine apparently blocked alpha(1H) and alpha(1I), but not alpha(1G) channels. Moreover, aranidipine blocked only alpha(1H) channels. By contrast, cilnidipine, felodipine, nifedipine and nitrendipine had little effects on these subtypes of T-type channels. The result indicates that the blockade of T-type Ca(2+) channels by derivatives of DHP Ca(2+) antagonist was selective for the channel subtype. Therefore, these selectivities of DHPs in blocking T-type Ca(2+) channel subtypes would provide useful pharmacological and clinical information on the mode of action of the drugs including side-effects and adverse effects.

Molecular characterization of T-type Ca(2+) channels responsible for low threshold spikes in hypothalamic paraventricular nucleus neurons.

The hypothalamic paraventricular nucleus (PVN) is composed of functionally heterogeneous cell groups, possessing distinct electrophysiological properties depending on their functional roles. Previously, T-type Ca(2+) dependent low-threshold spikes (LTS) have been demonstrated in various PVN neuronal types, including preautonomic cells. However, the molecular composition and functional properties of the underlying T-type Ca(2+) channels have not been characterized. In the present study, we combined single cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and patch-clamp recordings to identify subtypes of T-type Ca(2+) channels expressed in PVN cells displaying LTS (PVN-LTS), including identified preautonomic neurons. LTS appeared at the end of hyperpolarizing pulses either as long-lasting plateaus or as short-lasting depolarizing humps. LTS were mediated by rapidly activating and inactivating T-type Ca(2+) currents and were blocked by Ni(2+). Single cell RT-PCR and immunohistochemical studies revealed Cav3.1 (voltage-gated Ca(2+) channel) as the main channel subunit detected in PVN-LTS neurons. In conclusion, these data indicate that Cav3.1 is the major subtype of T-type Ca(2+) channel subunit that mediates T-type Ca(2+) dependent LTS in PVN neurons.

Changes in T-type calcium channel and its subtypes in overactive detrusor of the rats with partial bladder outflow obstruction.

AIMS: To investigate the activity of the T-type calcium channel (TCC) and the expression of its subtypes in overactive detrusor (OD) myocytes in rats after partial bladder outflow obstruction (PBOO). METHODS: Thirteen male Wistar rats with OD after PBOO (OD group) and eight sham-operated rats (control group) were studied. The two groups were compared regarding the expression of TCC subtype genes by reverse transcription-polymerase chain reaction (RT-PCR) and the TCC kinetics and cell action potential by whole-cell patch-clamp. RESULTS: The time course and density of the current were significantly higher in the OD cells than those in the control detrusor. Whole-cell patch-clamp analysis showed that the activation of TCCs in detrusor myocytes in the OD group was faster than the control group, but inactivation was almost the same in both groups, suggesting a significant enhancement of the Ca(2+) "window" current in the OD group. Patch-clamp recording of action potentials in the OD cells indicated an increase in excitability and a decrease in the repolarization interval. RT-PCR assay showed an abnormal expression of alpha1G subtype in the OD cells. CONCLUSIONS: TCCs could be one of the crucial factors for the abnormal excitation in OD cells. The development of OD after PBOO presumably relates to the increase in TCC current in the bladder cells, the enhancement of the Ca(2+) "window" current for Ca(2+) inflow, the prolongation of the intracellular calcium oscillations, and the acceleration of the cell depolarization.

Long-term and immediate effect of testosterone on single T-type calcium channel in neonatal rat cardiomyocytes.

In the cardiovascular system, T-type calcium channels play an important role for the intracellular calcium homeostasis and spontaneous pacemaker activity and are involved in the progression of structural heart diseases. Androgens influence the cardiovascular physiology and pathophysiology. However, their effect on native T-type calcium currents (I(Ca,T)) remains unclear. To test the chronic effect of testosterone on the cardiac I(Ca,T), cultured neonatal rat ventricular cardiomyocytes were treated with testosterone (1 nM-10 microM) for 24-30 h. Current measurements were performed after testosterone washout to exclude any acute testosterone effects. Testosterone (100 nm) pretreatment significantly increased whole-cell I(Ca,T) density from 1.26 +/- 0.48 pA/pF (n = 8) to 5.06 +/- 1.75 pA/pF (n = 7; P < 0.05) and accelerated beating rate. This was attributed to both increased expression levels of the pore-forming subunits Ca(v)3.1 and Ca(v)3.2 and increased T-type single-channel activity. On single-channel level, the increase of the ensemble average current by testosterone vs. time-matched controls was due to an increased availability (58.1 +/- 4.2 vs. 21.5 +/- 4.0%, P < 0.01) and open probability (2.78 +/- 0.29 vs. 0.85 +/- 0.23%, P < 0.01). Cotreatment with the selective testosterone receptor antagonist flutamide (10 mum) prevented these chronic testosterone-induced effects. Conversely, acute application of testosterone (10 microM) decreased T-type single-channel activity in testosterone pretreated cells by reducing the open probability (0.78 +/- 0.13 vs. 2.91 +/- 0.38%, P < 0.01), availability (23.6 +/- 3.3 vs. 57.6 +/- 4.5%, P < 0.01), and peak current (-20 +/- 4 vs. -58 +/- 4 fA, P < 0.01). Flutamide (10 microM) did not abolish the testosterone-induced acute block of T-type calcium channels. Our results indicate that long-term testosterone treatment increases, whereas acute testosterone decreases neonatal rat T-type calcium currents. These effects seem to be mediated by a genomic chronic stimulation and a nongenomic acute inhibitory action.

Diurnal gene expression patterns of T-type calcium channels and their modulation by ethanol.

The transient (T-type) calcium channel participates in the generation of normal brain rhythms as well as abnormal rhythms associated with a range of neurological disorders. There are three different isoforms of T-type channels and all are particularly enriched in the thalamus, which is involved in generating many of these rhythms. We report a novel means of T-type channel regulation in the thalamus that involves diurnal regulation of gene expression. Using real time polymerase chain reaction we detected a diurnal pattern of gene expression for all T-type channel transcripts. The peak of gene expression for the CaV3.1 transcript occurred close to the transition from active to inactive (sleep) states, while expression for both CaV3.2 and CaV3.3 peaked near the transition of inactive to active phase. We assessed the effect of chronic consumption of ethanol on these gene expression patterns by examining thalamic tissues of ethanol-consuming cohorts that were housed with the controls, but which received ethanol in the form of a liquid diet. Ethanol consumption resulted in a significant shift of peak gene expression of approximately 5 h for CaV3.2 toward the normally active phase of the mice, as well as increasing the overall gene expression levels by approximately 1.7-fold. Peak gene expression was significantly increased for both CaV3.2 and CaV3.3. Measurements of CaV3.3 protein expression reflected increases in gene expression due to ethanol. Our results illustrate a novel regulatory mechanism for T-type calcium channels that is consistent with their important role in generating thalamocortical sleep rhythms, and suggests that alterations in the pattern of gene expression of these channels could contribute to the disruption of normal sleep by ethanol.

The native T-type calcium current in relay neurons of the primate thalamus.

The generation of thalamic bursts depends upon calcium currents that flow through transiently open (T)-type calcium channels. In this study, we characterized the native T-type calcium current underlying thalamic burst responses in the macaque monkey. Current clamp recordings from lateral geniculate nucleus (LGN) slices showed characteristic burst responses when relay cells were depolarized from relatively hyperpolarized membrane potentials. These bursts could also be elicited by stimulation of excitatory synaptic inputs to LGN cells. Under voltage clamp conditions, the inactivation kinetics of native currents recorded from primate LGN neurons showed consistency with T-type currents recorded in other mammals and in expression systems. Real-time reverse transcriptase PCR performed on RNA isolated from the LGN (including tissues isolated from magnocellular and parvocellular laminae) detected voltage-dependent calcium channel (Ca(v)) 3.1, Ca(v) 3.2, and Ca(v) 3.3 channel transcripts. Ca(v) 3.1 occurred at relatively higher expression than other isoforms, consistent with in situ hybridization studies in rats, indicating that the molecular basis for burst firing in thalamocortical systems is an important conserved property of primate physiology. Since thalamic bursts have been observed during visual processing as well as in a number of CNS disorders, studies of the expression and modulation of these currents at multiple levels are critical for understanding their role in vision and for the discovery of new treatments for disruptions of thalamic rhythms.

The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors.

Recent studies have demonstrated a previously unrecognized contribution of T-type Ca2+ channels in peripheral sensory neurons to pain sensation (nociception). However, the cellular mechanisms underlying the functions of these channels in nociception are not known. Here, in both acutely dissociated and intact rat dorsal root ganglion neurons, we characterize a novel subpopulation of capsaicin- and isolectin B4-positive nociceptors that also expresses a high density of T-type Ca2+ currents. Using these "T-rich" cells as a model, we demonstrate that the endogenous reducing agent L-cysteine lowers the threshold for nociceptor excitability and induces burst firing by increasing the amplitude of T-type currents and shifting the gating parameters of T-type channels. These findings, which provide the first direct evidence of T-type Ca2+ channel involvement in the control of nociceptor excitability, suggest that endogenous T-type channel agonists may sensitize a unique subpopulation of peripheral nociceptors, consequently influencing pain processing under normal or pathological conditions.

Contrasting the effects of nifedipine on subtypes of endogenous and recombinant T-type Ca2+ channels.

There is evidence that nifedipine (Nif) - a dihydropyridine (DHP) Ca(2+)-channel antagonist mostly known for its L-type-specific action--is capable of blocking low voltage-activated (LVA or T-type) Ca(2+) channels as well. However, the discrimination by Nif of either various endogenous T-channel subtypes, evident from functional studies, or cloned Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 T-channel alpha 1 subunits have not been determined. Here, we investigated the effects of Nif on currents induced by Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 expression in Xenopus oocytes or HEK-293 cells (I(alpha 1G), I(alpha 1H) and I(alpha 1I), respectively) and two kinetically distinct, "fast" and "slow", LVA currents in thalamic neurons (I(LVA,f) and I(LVA,s)). At voltages of the maximums of respective currents the drug most potently blocked I(alpha 1H) (IC(50)=5 microM, max block 41%) followed by I(alpha 1G) (IC(50)=109 microM, 23%) and I(alpha 1I) (IC(50)=243 microM, 47%). The mechanism of blockade included interaction with Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 open and inactivated states. Nif blocked thalamic I(LVA,f) and I(LVA,s) with nearly equal potency (IC(50)=22 microM and 28 microM, respectively), but with different maximal inhibition (81% and 51%, respectively). We conclude that Ca(v)3.2 is the most sensitive to Nif, and that quantitative characteristics of drug action on T-type Ca(2+) channels depend on cellular system they are expressed in. Some common features in the voltage- and state-dependence of Nif action on endogenous and recombinant currents together with previous data on T-channel alpha 1 subunits mRNA expression patterns in the thalamus point to Ca(v)3.1 and Ca(v)3.3 as the major contributors to thalamic I(LVA,f) and I(LVA,s), respectively.

Contrasting anesthetic sensitivities of T-type Ca2+ channels of reticular thalamic neurons and recombinant Ca(v)3.3 channels.

Reticular thalamocortical neurons express a slowly inactivating T-type Ca(2+) current that is quite similar to that recorded from recombinant Ca(v)3.3b (alpha1Ib) channels. These neurons also express abundant Ca(v)3.3 mRNA, suggesting that it underlies the native current. Here, we test this hypothesis by comparing the anesthetic sensitivities of recombinant Ca(v)3.3b channels stably expressed in HEK 293 cells to native T channels in reticular thalamic neurons (nRT) from brain slices of young rats. Barbiturates completely blocked both Ca(v)3.3 and nRT currents, with pentobarbital being about twice more potent in blocking Ca(v)3.3 currents. Isoflurane had about the same potency in blocking Ca(v)3.3 and nRT currents, but enflurane, etomidate, propofol, and ethanol exhibited 2-4 fold higher potency in blocking nRT vs Ca(v)3.3 currents. Nitrous oxide (N(2)O; laughing gas) blocked completely nRT currents with IC(50) of 20%, but did not significantly affect Ca(v)3.3 currents at four-fold higher concentrations. In addition, we observed that in lower concentration, N(2)O reversibly increased nRT but not Ca(v)3.3 currents. In conclusion, contrasting anesthetic sensitivities of Ca(v)3.3 and nRT T-type Ca(2+) channels strongly suggest that different molecular structures of Ca(2+) channels give rise to slowly inactivating T-type Ca(2+) currents. Furthermore, effects of volatile anesthetics and ethanol on slowly inactivating T-type Ca(2+) channel variants may contribute to the clinical effects of these agents.

Pharmacology of recombinant low-voltage activated calcium channels.

Several types of voltage- or ligand-activated calcium channels contribute to the excitability of neuronal cells. Low-voltage-activated (LVA), T-type calcium channels are characterised by relatively negative threshold of activation and therefore they can generate low-threshold spikes, which are essential for burst firing. At least three different proteins form T-type calcium current in neurons: Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3. Expression of these proteins in various brain regions is complementary. Individual channel types could be distinguished by different sensitivity towards inorganic cations. This inhibition can contribute to the toxicity of some heavy metals. Selective inhibition of T-type calcium channels by organic blockers may have clinical importance in some forms of epilepsy. Mibefradil inhibits the expressed Ca(v2)3.1, Ca(v)3.2 and Ca(v)3.3 channels in nanomolar concentrations with Ca(v)3.3 channel having lowest affinity. The sensitivity of the expressed Ca(v)3.1 channel to the antiepileptic drugs, valproate and ethosuximide, is low. Ca(v)3.1 channel is moderately sensitive to phenytoin. The Ca(v)3.2 channel is sensitive to ethosuximide, amlodipine and amiloride. All three LVA calcium channels are moderately sensitive to active metabolites of methosuximide, i.e. alpha-methyl-alpha-phenylsuccinimide. Several neuroleptics inhibit all three LVA channels in clinically relevant concentrations. All three channels are also inhibited by the endogenous cannabinoid anandamide. A high affinity peptide blocker for these Ca channels is the scorpion toxin kurtoxin which inhibits the Ca(v)3.1 and Ca(v)3.2, but not the Ca(v)3.3 channel in nanomolar concentrations. Nitrous oxide selectively inhibits the Ca(v)3.2, but not the Ca(v)3.1 channel. The Ca(v)3.2, but not the Ca(v)3.1 channel is potentiated by stimulation of Ca(2+)/CaM-dependent protein kinase.

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.