© 2000 American Heart Association, Inc.
Editorials |
A Tale of Two (Calcium) Channels
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 joel.nargeot{at}igh.cnrs.fr
Key Words: Ca2+ channels • T-type channel • cardiac muscle
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Introduction |
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 Top Introduction References |
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Calcium 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 low-voltage-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 criss-crossing 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 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 µmol/L) versus 1G currents
(IC50>150 µmol/L).12 3
Preliminary data revealed a low Ni2+ sensitivity
(160 µmol/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 µmol/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 µmol/L
Ni2+ ions (IC50 16
µmol/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 excitation-contraction
coupling,24 but they could contribute to fine-tuning 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 tachycardia- induced 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 understanding of the physiological and pathological roles of LVA channel isoforms in cardiovascular cells.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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