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A Tale of Two (Calcium) Channels Joël Nargeot C Downloaded from http://circres.ahajournals.org/ by guest on August 10, 2017 properties of ICaT to those of the recombinant calcium currents generated by ␣1G and ␣1H expressed in human embryonic kidney cells. Most of the investigations are devoted to a comparison of the biophysical properties between ICaT from AT-1 cells and ␣1G/␣1H currents. The results indicate that most basic electrophysiological properties, such as current-voltage relationship and activation, inactivation, and deactivation properties, are rather similar and not discriminative. However, the recovery from inactivation of T-type currents, characterized by the sum of a fast and a slow time constant, is described as a functional signature of T-type channel isoform expression. The major difference between the two T-type channel isoforms relates to the relative amplitude of the slow recovery rate (s) as a strong functional criterion to establish a linkage between ICaT from AT-1 cells and ␣1G current. Such linkage is confirmed by reverse transcription–polymerase chain reaction (RT-PCR) experiments from cultured AT-1 cells using primers designed to amplify the III-IV loop of the three isoforms. The results show that only ␣1G, and predominantly a specific variant thereof, is revealed from sequencing of the subcloned PCR product corresponding to a single major band. The results presented on AT-1 cells are convincing, but it is reasonable to wonder whether they can be extended to any cardiac cell. Comparing the properties of native ICaT in cardiovascular cells with those of recombinant currents may be difficult because many studies have been conducted either under different experimental conditions or in different species. Cardiac T-type currents have often been studied in cultured embryonic or neonatal myocytes because of their low expression (or absence) in adult atrial (or ventricular) myocytes, except in several species such as chicken and guinea pig. Other studies report the characterization of cardiac ICaT induced by hormone treatment9 or pathology.10,11 On the other hand, T-type channel isoforms have also been cloned from different species, and the use of various external concentrations of calcium or barium ions to study their related currents sometimes confounds the comparisons between native and recombinant currents. Satin and Cribbs8 provide a comparison between the properties of the recombinant rat (␣1G) and human (␣1H) currents and ICaT from AT-1 (mouse) cells in a physiological calcium concentration. However, a large number of splice variants have been described for ␣1G in the different species3 and as discussed by the authors, it cannot be ruled out that splice variations in regions other than the III-IV loop may influence biophysical properties including recovery from inactivation. All of these splice variations involve connecting loops or the C-terminus. Curiously, nickel sensitivity was not investigated by Satin and Cribbs,8 even though it is considered an important assay to distinguish between the two isoforms because of the much higher Ni2⫹ sensitivity of ␣1H currents (IC50 5 mol/L) alcium influx through voltage-dependent calcium channels triggers excitation-contraction coupling and regulates pacemaking activity in the heart. Two distinct families of calcium channels have been identified in cardiac tissue: L-type calcium channels, which are essential in triggering Ca2⫹ release from internal stores, and lowvoltage-activated (LVA) T-type calcium channels (ICaT), whose role remains obscure in physiological and pathophysiological conditions. Functional features of ICaT include low threshold of activation, small unitary conductance, slow activation, and fast inactivation inducing a typical crisscrossing pattern of current traces for increasing depolarizations, negative steady-state inactivation, and slow deactivation kinetics. In addition, T-type currents compared with L-type currents are more sensitive to block by mibefradil and Ni2⫹ ions. Whereas L-type calcium channels have been extensively characterized at the functional and molecular levels, classical cloning strategies failed to identify an ␣1 subunit encoding for a T-type channel. An alternative approach to identifying new members of the calcium channel family used in silico cloning strategies with a search of genetic databases for sequences homologous but not identical to known Ca2⫹ channel ␣1 subunits.1 The identification of several expressed sequence tags and genomic sequences corresponding to a subset of distantly related ␣1 subunits resulted in the identification of full-length cDNAs encoding three distinct ␣1 subunits: ␣1G in rat,1 mouse,2 and human,3,4 ␣1H in human,5,6 and ␣1I in rat.7 Because ␣1H was obtained by screening a human heart library,5 it was originally considered the cardiac T-type channel isoform because of the presence in Northern blot analysis of a strong signal for ␣1H in the heart. However, an ␣1G transcript is also detected in adult heart, and it has become an interesting challenge to identify which isoforms underlie ICaT in cardiac cells. The article by Satin and Cribbs8 in this issue of Circulation Research aims to identify the ␣1 isoform encoding the ICaT in AT-1 cells, an immortalized cell line derived from mouse atrial tissue. The advantages of using this cell line after a short period of culture are the absence of sodium current and the relatively small amplitude of L-type current, allowing for a better isolation of ICaT. Satin and Cribbs have compared the The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Institut de Genetique Humaine, Montpellier, France. Correspondence to Joël Nargeot, Institut de Genetique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, 34396-Montpellier cedex 5, France. E-mail [email protected] (Circ Res. 2000;86:613-615.) © 2000 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org 613 614 Circulation Research March 31, 2000 Downloaded from http://circres.ahajournals.org/ by guest on August 10, 2017 versus ␣1G currents (IC50⬎150 mol/L).12,3 Preliminary data revealed a low Ni2⫹ sensitivity (160 mol/L) of cardiac T-currents in freshly dissociated atrial myocytes from neonatal rat cells, also suggesting a linkage with the ␣1G isoform.13 However, previous data in rabbit sinoatrial,14 adult guinea pig cells,15 and rat hypertrophic cells11 indicate that T-currents are totally blocked by a lower concentration of Ni2⫹ ions (about 40 mol/L). This would suggest either a differential expression of ␣1G and ␣1H isoforms among cardiac tissues, interspecies differences, or a possible developmental switch between isoforms. The latter hypothesis appears consistent with the results of Monteil et al,3 who observed in a dot-blot analysis a developmental regulation of human ␣1G transcripts with a prominent signal in embryonic compared with the adult heart. It must be emphasized, in agreement with such a hypothesis, that no ICaT has yet been recorded in adult human heart.16 Another study worth mentioning used an antisense strategy, which suggested that the cardiac T-type current in 3-week-old rats is related to the ␣1E subunit.17 This result remains puzzling because some properties such as threshold of activation and deactivation properties differ markedly between ICaT and ␣1E currents. In addition, ICaT from freshly dissociated neonatal rat atrial tissue was found13 to be insensitive to the ␣1E-specific toxin SNX 482. It is, however, worthwhile to note that T-type channel expression was induced by growth hormone treatment in the experiments of Piedras-Renteria et al.17 By contrast, with the absence of T-type current in adult human heart, an LVA calcium current was first reported in human atria,18 sharing some typical properties with sodium channels such as TTX sensitivity (designated ICaTTX). Its description in rat19 and guinea pig20 myocytes indicated that it is not exclusively observed in diseased human cells. The study of Heubach et al21 in this issue of Circulation Research demonstrates for the first time the coexistence of ICaTTX with ICaT in guinea pig ventricular myocytes. The results show that adult rat myocytes lack ICaT whereas adult guinea pig myocytes express both LVA currents that can be isolated by pharmacological dissection using TTX and Ni2⫹ in the absence of external sodium ions. As mentioned above, T-type currents are reported here to be blocked by 40 mol/L Ni2⫹ ions (IC50 16 mol/L), a concentration much lower than that required to block recombinant ␣1G currents but rather close to that reported to block ␣1H currents. There are major differences between the biophysical properties of ICaT and ICaTTX. ICaTTX exhibits a run-up after the rupture of the patch membrane, a lower voltage for peak current (10 mV), a more negative steady-state inactivation relationship, faster time constants for recovery from inactivation, and a faster rate of deactivation. Interestingly, ICaTTX was blocked by mibefradil, as is ICaT. The remaining question concerns the molecular basis of ICaTTX. Is this current related to modified sodium channels in the absence of external sodium ions20 or is it due to a new population of channels? Answering this will require the molecular identification of the pore-forming subunit using various strategies, including antisense, expression cloning, and other conventional molecular and biochemical isolation techniques. There is a lack of evidence for a correlation between ICaTTX and the controversial slip-mode conductance that has been described for sodium channels in the presence of protein kinase A (PKA) and cardiotonic steroids.22 The fact that ICaTTX does not require activation of the PKA pathway argues for a distinct population of channels. Additional experiments to test the effect of PKA inhibitors on ICaTTX would be of interest. Heubach et al21 underline some discrepancies between the TTX concentration required to block modified sodium channels and ICaTTX. In addition, the work of Lemaire et al18 on human atrial cells showed that ICaTTX similarly conducts calcium and barium ions. Permeation to barium ions was not tested on the modified sodium channels, but most of the conclusions were based on calcium transient measurements. This property seems atypical for sodium channels but might indicate either a strong alteration of the sodium channel permeability in the absence of extracellular sodium ions or molecular similarities between ICaT and ICaTTX. From the results presented in this issue of Circulation Research and other studies, it is now clear that two LVA channels (ICaT and ICaTTX) can coexist in cardiac myocytes. What is the physiological role of these LVA channels in the heart? T-type channels are assumed to play a role in pacemaking activity because of their presence in sinoatrial node and the negative chronotropic effect of Ni2⫹ ions.14 Whether ICaTTX is also expressed in sinoatrial cells is unknown, and it would also seem important to reinvestigate the role of LVA versus L-type calcium channels in the pacemaking activity. An interesting feature of T-type calcium currents is related to their slow deactivation kinetics and the existence of a window current in the range of the cell membrane resting potential. Slow deactivation can mediate larger calcium influx than high-voltage-activated channels during short depolarizations, as shown by the application of a neuronal-type action potential such as a voltage-clamp command on recombinant ␣1G channels,3 the calcium transients in response to different spike shapes or frequencies also being isoform dependent.23 In spite of their rapid kinetics of inactivation, the existence of a window current would confer a role of T-type channels in maintaining intracellular Ca2⫹concentration. T-type currents do not seem to play an important role in cardiac excitationcontraction coupling,24 but they could contribute to finetuning of basal calcium levels and control physiological processes such as hormone secretion, as previously suggested in adrenal cells.25 Their expression is also cell cycle dependent26 and their involvement in cell growth and proliferation was suggested in cardiac9 and smooth muscle cells27,28 respectively. In contrast to ICaT, ICaTTX is observed in human heart and is likely to be sensitive to mibefradil as found in guinea pig myocytes. Future studies will probably look for an overexpression of ICaT and ICaTTX in pathological conditions, which would be expected to generate cardiac arrhythmias. Interestingly, it was recently reported that mibefradil prevents tachycardiainduced electrophysiological remodeling in dogs.29 Thus, inhibition of both LVA currents by mibefradil might be considered in terms of pathophysiology. Combining specific functional assays and molecular tools should allow a better Nargeot understanding of the physiological and pathological roles of LVA channel isoforms in cardiovascular cells. References Downloaded from http://circres.ahajournals.org/ by guest on August 10, 2017 1. Perez-Reyes E, Cribbs LL, Daud A, Lacerda AE, Barclay J, Williamson MP, Fox M, Rees M, Lee J-H. Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature. 1998;391: 896 –900. 2. Klugbauer N, Marais E, Lacinova L, Hofmann F. A T-type calcium channel from mouse brain. Pflügers Arch. 1999;437:710 –715. 3. Monteil A, Chemin J, Bourinet E, Mennessier G, Lory P, Nargeot J. Molecular and functional properties of the human ␣1G subunit that forms T-type calcium channels. J Biol Chem. 2000;275:6090 – 6100. 4. Cribbs LL, Gomora JC, Daud AN, Lee J, Perez-Reyes E. Molecular cloning and functional expression of Ca(v)3.1c, a T-type calcium channel from human brain. 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KEY WORDS: Ca2⫹ channels 䡲 T-type channel 䡲 cardiac muscle A Tale of Two (Calcium) Channels Joël Nargeot Downloaded from http://circres.ahajournals.org/ by guest on August 10, 2017 Circ Res. 2000;86:613-615 doi: 10.1161/01.RES.86.6.613 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2000 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/86/6/613 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/