PHYSIOLOGY IN MEDICINE
In collaboration with
The American Physiological Society, Thomas E. Andreoli, MD, Editor
Calcium Currents and Arrhythmias:
Insights from Molecular Biology
Stephen R. Shorofsky, MD, PhD, and C. William Balke, MD
Calcium channels are critical to normal cardiac function. They
are involved in the generation and conduction of the action
potential and in contraction. Three surface membrane channels
have been identified. The L-type Ca channel is most abundant
and is responsible for Ca entry into the cell that triggers contrac-
tion. T-type Ca channels are most prevalent in the conduction
system and are probably involved in automaticity. A newly de-
scribed TTX-sensitive calcium current may be important in
“boosting” or enhancing conduction and contraction. The
main intracellular Ca channel resides in the sarcoplasmic retic-
C
alcium channels are wonderfully complex pro-
teins with vitally important roles in cardiac func-
tion. The opening of calcium (Ca) channels is in-
volved in pacemaker depolarization, supports conduc-
tion through the atrioventricular (AV) node, and
maintains the distinctive plateau of the cardiac action po-
tential. In addition, they serve as the primary gatekeepers
for Ca entry into cells, thus transducing the electrical sig-
nals at the surface of the myocyte into the biochemical
and mechanical events that result in a contraction. It is no
wonder, therefore, that Ca channels have attracted in-
tense interest over the last three decades.
Ion channels in the cardiac cell membrane are respon-
sible for the generation and control of the action poten-
tial. Thus, alterations in channel function often lead to
abnormal electrical activity and arrhythmias. In fact, ge-
netic defects in cardiac sodium and potassium channels
have been shown to cause the congenital long QT syn-
drome and its resultant arrhythmia, torsades de pointes
(1). To date, there has not been a specific arrhythmia that
results from an identified Ca channel defect in humans,
possibly because Ca channels have such a crucial role in
cardiac function that significant abnormalities in channel
function are lethal. This review, however, focuses on re-
From the Departments of Physiology and Medicine, University of
Maryland School of Medicine, Baltimore, Maryland.
Requests for reprints should be addressed to C. William Balke, De-
partment of Physiology, University of Maryland School of Medicine,
Howard Hall, Room 525, 660 West Redwood Street, Baltimore, Mary-
land 21201-1541.
䉷2001 by Excerpta Medica, Inc.
All rights reserved.
ulum and is responsible for the release of the Ca that activates
contraction. Oscillatory behavior of this channel influences the
sarcolemmal membrane, causing delayed aftercontractions and
arrhythmias such as those seen in digoxin toxicity. The on-go-
ing molecular characterization of these channels will enhance
our knowledge of their normal function and dysfunction in
disease states, leading to the development of new therapeutic
agents to treat arrhythmias and contractile dysfunction. Am J
Med. 2001;110:127–140. 䉷2001 by Excerpta Medica, Inc.
cent data concerning the molecular structure, function,
and regulation of these channels and identifies areas
where modulation of Ca channel function may have an
important role in the generation of arrhythmias and in
the progression and treatment of cardiac diseases.
NOMENCLATURE
Heart cells actually contain at least five types of Ca cur-
rents, three of which (L-type, T-type, and the channels
responsible for the newly described current (ICaTTX) are
expressed on the surface membrane, and two (the sarco-
plasmic reticulum Ca release channel and the IP3 recep-
tor) in internal membranes. The Table summarizes the
nomenclature, localization, and salient physiological
roles of each Ca channel type.
When clinicians speak of the Ca channel in the heart,
they usually mean the L-type Ca channel. This is not only
because of the fact that it exists in large quantities in myo-
cytes and was first recorded in 1967 in cardiac Purkinje
fibers (2), but also because these channels function as the
receptors for a number of clinically useful drugs, includ-
ing nifedipine, verapamil, and diltiazem.
This review concentrates on the L- and T-type Ca
channels, because they play the most obvious and prom-
inent role in cardiac arrhythmogenesis. The sarcoplasmic
reticulum (SR) Ca release channel, the IP3 receptor, and
the recently reported Ca-permeable Na channel (ICaTTX)
are reviewed briefly.
0002-9343/01/$–see front matter 127
PII S0002-9343(00)00586-6
 Calcium Currents and Arrhythmias/Shorofsky and Balke
Table 1. Cardiovascular Calcium Channels
Calcium Channel Type Location
L-type calcium channel Sarcolemma
T-type calcium channel Sarcolemma
ICa(TTX) channel Sarcolemma
Calcium release channel (ryanodine SR
receptor)
IP3 receptor SR
SR ⫽ sarcoplasmic reticulum.
L-TYPE CALCIUM CHANNELS
Physiological and Pathophysiological Roles
Sarcolemmal Ca channels constitute the major pathway
for Ca entry into the cell. The existence of a Ca-selective
current in heart muscle has been recognized for more
than 25 years (2). Two distinct types of channels with
high selectivity for Ca ions (L-type and T-type) are now
known to coexist in the surface membranes of heart cells
(3,4). The two types of channels can be distinguished on
the basis of their gating properties (ie, their pattern of
opening and its voltage dependence) and their pharma-
cological sensitivity. We first consider the “traditional”
Ca current of heart muscle, which is sensitive to modula-
tion by a variety of drugs. This current flowing through
so-called L-type channels (L for large and long lasting)
(4) is well characterized with respect to its role in cardiac
excitability, arrhythmogenesis, and excitation– contrac-
tion coupling. These channels are fairly plentiful, being
present in some 30,000 copies per ventricular myocyte
(5).
The L-type Ca channel is important in normal and
abnormal cardiac excitation. Ca current supports excita-
tion in the sinoatrial (SA) and atrioventricular (AV)
nodes as well as conduction through the AV node. Inter-
estingly, the AV node is the only site in the body where Ca
channels actually conduct an excitatory impulse; this dis-
tinctive feature, as well as use-dependent drug binding
explains why Ca channel blockers preferentially suppress
AV nodal conduction. In working heart muscle (atria and
ventricles) and in specialized conducting tissue, the prin-
cipal excitatory inward current underlying the action po-
tential plateau flows through L-type Ca channels. Every-
thing else remaining equal, an increase in Ca current pro-
longs depolarization and thereby increases the height and
duration of the action potential plateau. Conversely,
blockade of L-type channels shortens the action potential
in working myocardium and renders nodal tissue inex-
citable.
128 February 1, 2001 THE AMERICAN JOURNAL OF MEDICINE威
Ligands Physiological Roles
Dihydropyridines Atrioventricular nodal conduction
Benzothiazepines Slow response upstroke
Phenylalkylamines Excitation–contraction coupling
Plateau depolarization
Mibefradil Pacemaker depolarization
Cell growth and differentiation?
Tetrodotoxin Augmenting depolarization
Excitation–contraction coupling?
Ryanodine SR calcium release
Inositol triphosphate Modulation of SR Ca release ?
Other functions
L-type Ca channels also play potentially important
roles in arrhythmia generation and their treatment. Ca
channels are capable of mediating the inward current that
underlies some types of early afterdepolarizations
(EADs) that cause triggered arrhythmias and torsades de
pointes in animals (6,7). As mentioned earlier, there has
not been a demonstration in humans of a Ca channel
defect causing torsades de pointes, or any other arrhyth-
mia thought to be the result of EADs. Ca channels can
also support slow conduction in reentrant circuits, espe-
cially in depolarized tissue, although this mechanism ap-
pears to predominate in only a small minority of patients
with inducible ventricular tachycardia. By their effects on
automaticity and AV node conduction, L-type Ca chan-
nels may be involved in the generation of certain auto-
matic arrhythmias [eg, repetitive monomorphic right
ventricular outflow tachycardia (RVOT) and some atrial
tachycardias], and modulation of these Ca channels is
useful in treating many supraventricular tachycardias
(eg, AV nodal reentry, atrial fibrillation, and atrial flut-
ter).
An appreciation of the role of Ca channels in excita-
tion– contraction coupling is important in understand-
ing the potential side effects of Ca channel blockers.
Ringer (8) first realized in 1883 that Ca ions must be
present in the extracellular medium for the heart to gen-
erate force. Even though the amount of Ca entering the
cell during a single action potential through L-type chan-
nels appears to be too small to account fully for contrac-
tile activation (9), Ca influx through these channels is a
necessary prerequisite for the initiation of contraction
(10 –12).
Several organic L-type channel inhibitors exist. The
classic members of this drug family come from three
chemical groups: the phenylalkylamines (eg, verapamil),
the benzothiazepines (eg, diltiazem), and dihydropyri-
dines (eg, nifedipine, nitrendipine, and amlodipine) (13).
Exposure to any of the Ca channel antagonists can abolish
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 Calcium Currents and Arrhythmias/Shorofsky and Balke

Figure 1. Schematic model of the transmembrane topology of the ␣1 subunit of the L-type Ca channel. The P loops in each repeat
are indicated by the thick lines. [Reproduced with permission from W.B. Saunders Company, Cardiac Electrophysiology, 3rd ed.
(147).]

contractile force in isolated myocardial preparations by
interrupting the flux of Ca into the cell by means of L-type
Ca channels. There is good evidence that the high-affinity
binding sites for these compounds are located on the ␣1
subunit of the L-type Ca channel (14), the structure of
which is discussed below.
Structure–function Relationship
Channels are pore-forming proteins that open and close
in response to defined stimuli. The processes whereby
channels open and close are collectively known as gating,
whereas permeation describes how a given channel con-
ducts ions when it is open. The conceptual distinction
between gating on the one hand and permeation on the
other predates the molecular biology era. Now that many
channels have been cloned, expressed, and mutated, spe-
cific and separate structures have been identified that me-
diate gating and permeation. Among biological voltage-
dependent ion channels, those selective for potassium (K
channels) are the smallest and, not coincidentally, the
most thoroughly characterized (15). Despite the fact that
channels selective for Na or Ca are larger and more com-
plex than K channels, several prominent features have
emerged.
L-type Ca channels were first purified from skeletal
muscle and consist of five subunits: ␣1 (165 kD), ␣2 (130
kD) linked to the ␦ subunit (28 kD) by a disulfide bond, ␤
(55 kD), and ␥ (32 kD) (16,17). The primary sequence of
the ␣1 subunit was then determined from the cDNA se-
quence encoding the protein in rabbit skeletal muscle
(14). Remarkable homology exists between the ␣1 sub-
unit of the Ca channel and the pore-forming (␣) subunit
of voltage-dependent Na channels, both having four ho-
mologous domains composed of six transmembrane seg-
ments each (16). Figure 1 shows a schematic diagram of
the ␣1 subunit of the L-type Ca channel (the presumed
pattern of assembly with the other subunits is dia-
grammed in Figure 2). The overall protein, deduced from
the cDNAs of various isoforms cloned to date (including
that from heart), is more than 2,000 amino acids long and
consists of four internally homologous domains (I to IV).
Each domain is believed to span the membrane six times
(S1 to S6). The fourth such segment (S4) in each domain
is distinguished by the repetition, at every third position,
of positively charged amino acids (lysine or arginine)
(16,18). Because Na and Ca channels open in response to
depolarization, elucidation of the S4 structure immedi-
ately suggested a physical correlate for the voltage sensor
involved in activation gating. Site-directed mutagenesis
to modify the net charge in S4 in Na channels alters the
voltage dependence of activation, verifying the prediction
that S4 functions as a voltage sensor (19). The ␣1 subunit
contains the binding sites for Ca channel blocking drugs,
including the dihydropyridines, benzothiazepines, and
phenylalkylamines (14). The cloned ␣1 subunit can be
expressed in Xenopus oocytes, dysgenic myotubes, or
mammalian tissue culture cells and is capable of conduct-
ing Ca currents (20 –22). These studies established that
the fundamental properties of the channel allowing for
selective permeation of Ca across the membrane reside
entirely in the ␣1 subunit. Beyond these basic observa-
tions, much remains to be learned about the specific sites
in the L-type Ca channel that underlie its distinctive gat-
ing and permeation properties. Expression of chimeric ␣1
constructs in dysgenic mouse muscle has revealed that
the loop between domains II and III is important in iso-
form-specific excitation– contraction coupling (22),
whereas domain I confers features of activation gating
(23).

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 Calcium Currents and Arrhythmias/Shorofsky and Balke

Figure 2. Schematic representation of the regulatory subunits and their putative relationship to the ␣1 subunit of the L-type Ca
channel. [Reproduced with permission from W.B. Saunders Company, Cardiac Electrophysiology, 3rd ed. (147).]

Na and Ca channels enter a long-lived nonconducting
state during maintained depolarization, a gating process
known as “inactivation” (24,25), which is not altered by
changes in S4 (19). Unlike S4, no feature of Na or Ca
channels suggests a role in inactivation merely from in-
spection of the primary sequence. Nevertheless, mu-
tagenesis has revealed that the cytoplasmic linker be-
tween domains III and IV, which has been highly con-
served during evolution, forms at least part of the
inactivation gate of Na channels (19). Thus, two regions
of the Na channel are clearly implicated in gating: S4
senses voltage and initiates activation gating, and the
III–IV linker figures prominently in inactivation. Muta-
tions in either region do not grossly influence pore prop-
erties. The functional roles of S4 and of the III-IV linker in
the Ca channel have yet to be investigated, partly because
the Ca channel clones have not been available as long as
those for the Na channel, but also because Ca channels
have proved to be considerably more difficult to express
in surrogate cells. Nevertheless, the fact that Na channels
are so similar in their overall molecular architecture gives
good reason to predict the likely functions of the homol-
ogous regions of the Ca channel.
A confluence of biophysical and molecular biological
data has defined regions and residues in the Na channel ␣
subunit that are important in forming the permeation
pathway. A region of striking sequence conservation oc-
curs between the fifth and sixth membrane-spanning re-
gions (S5 to S6), also called the P loop (P for pore or
permeation) (26). There is now solid evidence, from elec-
trophysiological analysis of mutant Na and K channels,
that the P loops from each of the four domains come
together in the center of the protein to line the pore
(15,27). The P loops have been proposed to form ␤-hair-
pins that form a ␤-barrel structure, but the precise con-
formation is not yet known.
Ca channels are very selective. Typically, the perme-
ability of the channel to Ca is a thousand-fold higher than
the permeability to K or Na (28). Yet, a single Ca channel
can conduct more than 106 Ca ions per second across the
cell membrane (29). To explain how a Ca channel recon-
ciles the conflicting demands of maintaining a large con-
ductance in the face of exquisite selectivity, models have
been proposed in which Ca binds with high affinity at two
or more sites down the permeation pathway (30,31).
High-affinity binding makes the channel selective, while

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 Calcium Currents and Arrhythmias/Shorofsky and Balke
the presence of multiple binding sites enables a high
throughput rate: simultaneous occupancy by two or
more Ca ions accelerates flux resulting from electrostatic
ion–ion repulsion. The model handily explains the ability
of Ca channels to conduct large fluxes of monovalent
cations when divalent cations are absent (30,31). Re-
markably, this model and others (32) were proposed
without any knowledge of the actual protein sequence of
any voltage-dependent ion channels.
The P loops from Na and Ca channels are largely con-
served but differ in the presence of acidic residues in re-
peats III and IV. Substituting acidic residues in these po-
sitions of the Na channel produces the essential features
of permeation in Ca channels (monovalent permeability
at very low [Ca], Ca selectivity at higher [Ca]) without
grossly changing other properties of the channel, such as
gating kinetics (33). While confirming that the S5–S6 re-
gion in each repeat of the Na channel is an integral part of
the permeation pathway, these results speak only indi-
rectly to the question of how the Ca channel itself forms
the pore. More direct confirmation for the role of S5–S6
in Ca channel permeation comes from recent mutagene-
sis studies that suggest that a ring of highly conserved
glutamate residues in the P loops confers Ca selectivity
(34 –36).
Dihydropyridine (DHP) Ca channel ligands bind spe-
cifically to L-type Ca channels and, like local anesthetics
(LA) acting on Na channels, do so in a voltage-dependent
manner. Bean (37) demonstrated that voltage protocols
that favor Ca channel inactivation markedly enhance the
potency of channel block by nitrendipine; he invoked the
modulated receptor hypothesis to explain the results,
postulating a 3,000-fold higher affinity for the inactivated
state than for the resting state. In agreement with this
interpretation, Kamp and Miller (38,39) showed that ra-
diolabeled DHPs bind much more avidly during surface
membrane depolarization, either in intact cells or in
membrane vesicles. Numerous other studies have
pointed out the importance of the gating-related channel
state in the binding not only of DHPs but also of benzo-
thiazepines and phenylalkylamines to L-type Ca channels
(40,41). Virtually nothing is known about the structural
basis for the interaction; particularly intriguing is the fact
that photoaffinity-labeling experiments place the DHP
binding site at the external face of the channel (in contact
with III-S5–S6 and with the external end of IV-S6), far
from any putative intracellular inactivation gate (14).
Regulation
Beta-adrenergic stimulation. Sympathetic stimulation
causes an increase in heart rate and augmented contrac-
tility. L-type Ca channels figure prominently in mediat-
ing these effects of ␤-adrenergic stimulation (42– 44).
When an agonist binds to the cardiac ␤ receptors, a series
of events takes place. The occupied ␤ receptor activates a
February 1, 2001
second membrane protein called G protein (for its ability
to bind and to split guanosine triphosphate [GTP]). The
activated ␣ subunit of the G protein in turn stimulates the
membrane-bound enzyme adenylate cyclase. Biological
membranes contain several types of G proteins (45,46).
The type that mediates stimulation of adenylate cyclase
(Gs) carries the subscript s to distinguish it from others of
inhibitory (Gi) or unrelated (Go) function. The enzyme
adenylate cyclase catalyzes the formation of cyclic aden-
osine 3⬘,5⬘-monophosphate (cAMP) from ATP, tending
to increase the cellular content of cAMP (43). Cyclic AMP
regulates a number of cellular functions by promoting
selective protein phosphorylation, initiated by the bind-
ing of cAMP to the regulatory subunit of cAMP-depen-
dent protein kinase (protein kinase A). The binding of
cAMP liberates the catalytic subunit of the enzyme, which
then phosphorylates a variety of cellular proteins (47).
The cardiac L-type Ca channel appears to be a particu-
larly important substrate for phosphorylation by protein
kinase A, based on evidence primarily from functional
studies (44,48,49).
Ca channels, when phosphorylated by ␤-adrenergic
stimulation, are considerably more active than in the
nonphosphorylated state, thus producing an increase in
the Ca current during each action potential (44,48,50).
An increase in Ca current can occur by several different
means when one examines the underlying single-channel
events. Macroscopic current (I) is the product of the
number of channels in the membrane available for open-
ing (N), the probability that a channel will open (Po), and
the current carried by a single, open channel at that volt-
age (i), or I ⫽ N 䡠 Po 䡠 i. Activation of the cAMP-depen-
dent phosphorylation pathway results in an increase in
channel availability (seen as an increase in the number of
openings during a depolarizing pulse) and an increase in
the probability of opening. The latter is manifested both
as an increased frequency of openings during a depolar-
izing pulse (51) and as an augmentation of the number of
ultralong openings (52). Under control conditions, sin-
gle-channel openings are quite brief and often do not
occur at all during depolarizations to various test poten-
tials. In contrast, exposure to 1 ␮M isoproterenol (a
␤-adrenergic agonist) increases channel availability (as
evidenced by a reduction in the number of blank sweeps)
and increases the occurrence of a long-opening mode of
the channel.
Thus, a complex chain of molecular events produces
an increase in L-type Ca current and ultimately enhanced
Ca delivery to the cytoplasm. The increase in Ca current
increases conduction through the AV node and increases
the firing rate of normal and subsidiary pacemakers. Ab-
normal slow conduction will be accelerated to the extent
that it is mediated by Ca channels, thus altering the like-
lihood of reentry.
Recently it has become clear that G proteins can di-
THE AMERICAN JOURNAL OF MEDICINE威 Volume 110 131
 Calcium Currents and Arrhythmias/Shorofsky and Balke
rectly influence the activity of ion channels, even in the
absence of second messengers, such as cAMP. Exposure
to a purified preparation of activated Gs ␣ subunits in-
creases the likelihood that L-type Ca channels will open in
bilayers (53). Although such cell-free experiments are
consistent with the idea that part of the ␤-adrenergic
stimulation of Ca channels is mediated directly by G pro-
teins, the fact that cAMP and its derivatives mimic the
effects of ␤ agonists on Ca currents in intact cells implies
that G proteins are not required. Nevertheless, the recog-
nition of direct ion channel gating by G proteins opens
the novel possibility of drugs that might act on this G
protein link, rather than on the more conventional sec-
ond-messenger–mediated cascade.
Regulation by intracellular magnesium and Mg-ATP.
Although often taken for granted, the importance of
magnesium (Mg) in the control of cell physiology cannot
be overemphasized. Whether the cell is using energy sub-
strates, pumping up or dissipating ion gradients across a
membrane, contracting, or synthesizing nucleic acids and
proteins, Mg participates in the process. The vital re-
quirement for Mg and Mg-nucleotide complexes in sup-
porting enzyme activity has long been recognized. More
recently, a growing body of work has elucidated a number
of mechanisms whereby ion channels can sense and re-
spond to Mg and Mg nucleotides.
L-type Ca channels are subject to several modulatory
actions of Mg. Direct channel blocking effects of both
extracellular and intracellular Mg have been observed
(54). Furthermore, the rate of inactivation of inward Ba
or Na currents through L-type Ca channels has been re-
ported to increase, and the amplitude of the currents to
decrease, when intracellular Mg was raised from 0.3 to 3
mM in frog cardiomyocytes (55). When Ca is the charge
carrier, raising intracellular Mg also reduces the ampli-
tude of Ca current in guinea pig cardiomyocytes (56). In
the frog, this effect is accentuated by prior channel phos-
phorylation, perhaps by altering the affinity of the Mg
binding site (55).
Although the role of Mg-ATP in the cAMP-mediated
phosphorylation of Ca channels has been recognized, rel-
atively little attention has been paid to investigating the
effects of Mg-ATP itself on L-type Ca channels. Previous
studies have shown that the inclusion of Mg-ATP in suf-
ficient quantities in intracellular solutions can retard the
rate of “rundown” of Ca current (57). In addition, a pos-
itive correlation between intracellular Mg-ATP and the
amplitude of cardiac L-type Ca currents has been re-
ported (58). Both these effects have been assumed to be
related to the importance of Mg-ATP as a substrate for
phosphorylation.
Exploiting the Mg binding properties of the photosen-
sitive compounds [DM-nitrophen (59) and caged Mg],
O’Rourke et al (60) compared the effects of various intra-
132 February 1, 2001 THE AMERICAN JOURNAL OF MEDICINE威
cellular concentrations of Mg-ATP on L-type Ca currents
with the predominant mechanism for regulating Ca
channels, ␤-adrenergic receptor activation, and cAMP-
dependent phosphorylation. In a series of elegant studies,
they demonstrated that a direct relationship exists be-
tween intracellular Mg-ATP levels and L-type Ca current
amplitude, which was independent of phosphorylation.
Elucidation of the phosphorylation-independent
pathway does not diminish the importance of cAMP-de-
pendent phosphorylation as the primary mechanism for
increasing Ca channel activity in the heart. However, it
does stimulate speculation as to the supplementary role
of Mg-ATP in linking metabolism to Ca influx. As
pointed out by Goldhaber et al (61), cytosolic Ca tran-
sients are reduced in ventricular myocytes during meta-
bolic inhibition as a result of both a reduction in L-type
Ca current and impaired coupling between the Ca cur-
rent and SR Ca release, even though the caffeine-releas-
able pool of Ca is actually greater than in the control
condition. Similarly, Taniguchi et al (62) reported that
Ca current depression induced by metabolic inhibition
could be reversed by injection of ATP. During times of
metabolic stress, the ability of channels to sense Mg-ATP
levels may serve as a protective mechanism to reduce the
metabolic demand associated with Ca removal by cutting
back on the trigger for Ca release. This type of stopgap
regulation would be carried out at no energy cost to the
cell and would function even when the phosphorylation
pathway is in high gear. One situation in which this may
occur is during ischemia, when cytosolic Mg increases,
Mg-ATP decreases (63), and the sympathetic nerves are
in overdrive. The overall picture of ion channel activity at
this time may include inhibition of the SR Ca release
channels (64,65), activation of ATP-sensitive K channels,
and perhaps a reduction in Ca current as a result of both
the decrease in Mg-ATP and the increase in Mg.
T-TYPE CALCIUM CHANNELS
Physiological and Pathophysiological Roles
T-type Ca channels are found in cardiac and vascular
smooth muscle. They can be distinguished from L-type
channels on the basis of their distinctive biophysical char-
acteristics (gating and permeation properties), relative
distribution, differential pharmacology, and structure
(66). Compared with L-type channels, T-type channels
open at significantly more negative membrane potentials
that are near the resting potential, rapidly inactivate
(hence, T for transient), slowly deactivate, and have a low
conductance (hence, T for tiny) (3,4,66).
In the heart, T-type Ca channels are expressed in high-
density in SA and AV nodal tissue (67) and Purkinje cells
(68), and are relatively sparse or absent in most atrial and
ventricular cells. This distribution is consistent with their
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 Calcium Currents and Arrhythmias/Shorofsky and Balke
putative role in pacemaker impulse formation. T-type
channels probably do not contribute to the action poten-
tial upstroke of cardiac ventricular cells, because their
relatively small and transient current component is dom-
inated by the coincident and much larger classical Na
current (69). However, T-type channels may play a role in
action potential generation, particularly in cell types that
lack a high density of Na channels, such as vascular
smooth muscle (70).
T-type channels have been implicated in cell growth
under both physiological and pathophysiological condi-
tions. T-type channels are abundant in the heart during
fetal development (71) and during rapid growth in the
early postnatal period (72). In addition, cardiac T-type
Ca channel density is increased under several conditions
that are associated with myocyte growth, including rats
with growth-hormone–secreting tumors (73), ventricu-
lar cells from failing cardiomyopathic hamsters (74), and
endothelin-1 induced hypertrophy in neonatal rat ven-
tricular cells (75). Experimentally induced pressure over-
load hypertrophy in cats induced a re-expression of T-
type Ca channels in ventricular cells that normally do not
have any demonstrable T-type Ca current (76). Further-
more, pretreatment with a selective inhibitor of T-type
channels, mibefradil, reduces intimal proliferation after
experimentally induced vascular injury in rats (77). Of
interest, long-term treatment with mibefradil in a rat
model of chronic heart failure was associated with an in-
crease in survival (78). Although the precise mechanism
for this response is as yet unknown, the reappearance of
the fetal phenotype may be a marker for the transition of
cardiac hypertrophy to heart failure and may contribute
to the increased incidence of sudden death and pump
failure characteristic of the failing heart.
Compared with the well-studied L-type Ca channel,
the pharmacology of the T-type channels is less well char-
acterized. Several drugs and inorganic compounds affect
T-type channels, including amiloride (79) and its deriva-
tive 3,4-dichlorobenzamil (80), verapamil, diltiazem
(81), flunarizine (81), and tetradrine. Nickel and cad-
mium also block T-type channels. However, all of these
agents also act on L-type Ca channels and, therefore, are
not useful for dissecting out the effects of selective T-type
channel modulation on cardiac electrical or mechanical
activity. Recently, a novel Ca channel antagonist, mibe-
fradil, has been shown to inhibit both L- and T-type
channels with a marked selectivity for the T-type channel
(82– 85). It preferentially affects the coronary vasculature
relative to the myocardium. In contrast to other Ca chan-
nel antagonists, its vasodilatory effects are not associated
with negative inotropic or conduction abnormalities, but
it did cause sinus slowing. Although its clinical utility has
been compromised by serious drug interactions (86), it
remains potentially useful as a research tool for a detailed
characterization of the contribution that T-type Ca chan-
February 1, 2001
nels may make to cardiac electrical and mechanical func-
tion.
Structure-function Relationship
Until recently, information regarding the structure of T-
type Ca channels was inferential and indirect. Augmen-
tation of T-type current by exposure to neuraminidase
(87) suggested that the molecules are glycosylated and
that such glycosylation might modulate function. Other
structural features were implied from the comparison
with L-type channels. Despite important differences in
single-channel conductance (T-type channels are about
one third to one half as large as L-type channels) (88),
their selectivity for Ca over monovalents is comparable,
and both types of channel can conduct monovalent cat-
ions well when divalent cations are absent (88). On the
other hand, T-type channels do not exhibit differences in
inactivation depending on the ion that carries the charge,
unlike L-type channels, which display prominent Ca-me-
diated inactivation (4,88). Finally, an important struc-
tural difference in the outer channel mouth regions is
suggested by the fact that T-type channels do not bind
dihydropyridines (3,4).
Perez-Reyes et al (89) recently cloned the first member
(␣1G) of a new family of Ca channels in rat, mouse, and
human neuronal tissue that is distinct from all previously
cloned Ca channels. They localized the gene encoding
human ␣1G to human chromosome 17q22 and the
mouse locus to distal mouse chromosome 11. Functional
expression studies in Xenopus oocytes provided compel-
ling evidence that ␣1G was, indeed, a T-type Ca channel
on the basis of its negative voltage range of activation, fast
inactivation, slow deactivation, and tiny unitary conduc-
tance of approximately 7.5 pS. On the heels of this land-
mark report, Perez-Reyes et al cloned a second member
of this family of T-type Ca channels (␣1H) from human
heart (90). The cDNA of ␣1H was consistent with the
general structure of a voltage-gated Ca channel, and there
was a high degree of homology with human and mouse
␣1G. Specifically, both ␣1H and ␣1G are predicted to
have a four-domain structure with conserved pore loops
and voltage sensors. In addition, ␣1H lacks both an iden-
tifiable motif for ␤ subunit binding and a Ca binding
domain that would confer Ca-dependent inactivation. In
contrast to human ␣1G, human ␣1H was mapped to a
different chromosome, human chromosome 16p13.3.
Both ␣1G and ␣1H were found in brain and heart. How-
ever, ␣1G was most abundant in brain, and ␣1H was
most abundant in heart. Importantly, heterologous ex-
pression of ␣1H in HEK cells produced Ca currents with
the biophysical hallmarks of native T-type channels in-
cluding a single-channel conductance for barium of ap-
proximately 5.3 pS and a sensitivity to mibefradil at the
concentration reported for native T-type Ca channels.
The identification of multiple genes encoding T-type Ca
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 Calcium Currents and Arrhythmias/Shorofsky and Balke
channels represents a major advance in the study of volt-
age-gated ion channels. The availability of recombinant
T-type channels provides the tools necessary to ade-
quately separate the contributions of T- and L-type Ca
channels to cardiac function and to identify T-type Ca
channel–related pathophysiology.
Regulation
T-type Ca channels are not prominently regulated by
␤-adrenergic mechanisms (91) but do exhibit a promi-
nent up-regulation with norepinephrine (92), phenyl-
ephrine (an ␣-adrenergic agonist) (93), and extracellular
ATP (94). Endothelin has also been found to augment Ca
entry through T-type channels in heart cells (75). These
agents only modestly alter L-type currents. Developmen-
tal changes in the relative densities of L-type and T-type
channel expression have been deduced (94,95) and may
turn out to be clinically important in distinguishing
mechanisms of pediatric versus adult automaticity. In the
cultured chick myocyte model, developmentally young
cells exhibit large T-type currents (94). The mammalian
correlates of these changes remain to be fully defined.
Interestingly, Nuss and Houser (76) have observed that
T-type currents are enhanced in hypertrophic ventricular
cells from the cat, and this may represent an electrophys-
iological example of the general truism that cardiac hy-
pertrophy turns back the developmental clock by activat-
ing a “program” of fetal gene expression.
TETRODOTOXIN-SENSITIVE CALCIUM
CHANNELS (ICa(TTX))
Recently, a new and functionally distinct inward current
component has been identified in rat ventricular cells
(96). The channels that carry this current are permeable
to both Na and Ca and are blocked by tetrodotoxin
(TTX), hence their designation as ICa(TTX). This new
component displays different kinetics, different voltage
ranges for both activation and inactivation, and different
permeability properties from classical sodium channels
and L- and T-type Ca channels. Specifically, ICa(TTX) ac-
tivates over a more negative voltage range than classical
Na and T-type channels. Therefore, it may act to amplify
the depolarization delivered by Purkinje fibers or adja-
cent ventricular cells and so provide the immediate trig-
ger for the generation of the cardiac action potential. To
the extent that ICa(TTX) triggers the cardiac action poten-
tial, it may be important for cardiac arrhythmias and
their control. In addition, pharmacological modulation
of ICa(TTX) would be predicted to affect the timing and
conduction of the cardiac action potential without com-
promising the action potential itself (ie, Vmax or over-
shoot potential). Consequently, this pharmacological
strategy would be expected to have fewer and less severe
side effects compared with traditional antiarrhythmic
134 February 1, 2001 THE AMERICAN JOURNAL OF MEDICINE威
agents that are targeted to the classical cardiac Na chan-
nel.
Because ICa(TTX) is permeable to Ca as well as Na, it
may also play a role in providing the Ca trigger for SR Ca
release by one of two possible mechanisms. 1) Na perme-
ation of ICa(TTX) could elevate the concentration of intra-
cellular Na in a “restricted space” near the sarcolemmal
membrane and thereby activate Ca influx by means of the
Na/Ca exchanger (ie, “reverse mode”) to a level that con-
tributes to the trigger for SR Ca release. 2) Ca permeation
of ICa(TTX) could contribute directly to the trigger for SR
Ca release.
Apart from this initial characterization and the dem-
onstration that the current is augmented by ␤-adrenergic
stimulation (96,97), no information is yet available re-
garding the relative permeabilities, structure, and regula-
tion of ICa(TTX). Importantly, ICa(TTX) may represent a
distinct new Na (or Ca) channel isoform or a differen-
tially regulated or otherwise posttranslationally modified
Na (or Ca) channel.
SARCOPLASMIC RETICULUM CALCIUM
RELEASE CHANNELS (RYANODINE
RECEPTORS)
Physiological and Pathophysiological Roles
The Ca release channels of the sarcoplasmic reticulum
(SR) are entirely intracellular; they mediate the rapid ef-
flux of Ca from the SR lumen into the cytosol during each
cardiac cycle. In the scientific literature, SR Ca release
channels are often called ryanodine receptors because
they bind the plant-derived toxin ryanodine with nano-
molar affinity.
The SR has been generally accepted as the predominant
source of activator Ca for activation of the myofilaments
and contraction in both skeletal and cardiac muscle
(98,99). However, ultrastructural comparison with skel-
etal muscle reveals that cardiac SR occupies a much
smaller fraction of the intracellular volume (100). Fur-
thermore, the mechanism of signal transduction cou-
pling the cell membrane and the terminal cisternae of the
SR is still incompletely understood. As complicating fac-
tors, substantial differences have been reported regarding
the contribution of SR Ca in atrial as opposed to ventric-
ular tissue, between mammalian and lower vertebrate
hearts, and even among ventricles from different mam-
malian species (101).
Structure–function Relationship
Ca release channel proteins are the largest biological
channels yet described; they are so large that the shadows
of their cytoplasmic protrusions can be visualized in rou-
tine electron micrographs as the “foot” processes that
span the gap between the SR and surface membranes. In
contrast to membrane structures embedded in the sarco-
Volume 110
 Calcium Currents and Arrhythmias/Shorofsky and Balke
lemma, functional proteins located in the SR are often
studied under experimental conditions where the integ-
rity of the cell has to be disrupted by “skinning” myocar-
dial cells or by preparing vesicular membrane fractions
from the SR. At the intact cellular level, several agents that
inhibit SR Ca release markedly reduce contractility, and
drugs that facilitate SR Ca release activate contraction.
Functional studies (10,11) suggest that in the intact cell,
Ca release from the SR is triggered by the increase of
intracellular free Ca concentration ([Ca]) initially pro-
duced by the L-type Ca current. Such observations sup-
port the mechanism of Ca-induced Ca release from the
SR, first described in skinned preparations (102). Several
recent studies have advanced our understanding of the
quantitative relationship (or the gain) between the trigger
Ca provided by the L-type Ca current and activator Ca
provided by the SR Ca release channel (103,104). They
have shown that SR Ca release is controlled by events
localized to the region of the L-type Ca channels and SR
Ca release channels (ryanodine receptors), and these
events may be very different from those seen macroscop-
ically in the whole cell.
Several recent experimental studies provide support
for this local coupling of the L-type Ca channel and the SR
Ca release channel. 1) Immunocytochemical and ultra-
structural studies demonstrate the close physical proxim-
ity and clustering of L-type Ca channels and ryanodine
receptors. In ventricular cells, L-type Ca channels were
closely associated with ryanodine receptors at the T tu-
bules (105). In atrial cells, which lack T tubules, L-type Ca
channels and ryanodine receptors also co-localized to the
sarcolemma. 2) Spontaneous, spatially localized non-
propagating transient elevations in [Ca] (ie, Ca sparks)
have been observed in resting single cardiac cells (106)
and in intact cardiac muscle (107). 3) López-López et al
(108,109) and others (110,111) have shown that SR Ca
release was controlled locally (ie, Ca sparks) by L-type Ca
current in voltage-clamped rat ventricular cells. Ca sparks
evoked by membrane depolarization behave in a stochas-
tic manner (108 –110) and have a voltage and time de-
pendence similar to that of the L-type Ca channel (109).
4) Ca sparks do not appear to be elicited by Ca entry by
means of the reverse mode of the Na/Ca exchanger under
certain experimental conditions (109). 5) Consistent
with the immunocytochemical and ultrastructural stud-
ies, Ca sparks do not occur randomly throughout the cell
but localize to the region of the T tubule and the junc-
tional SR (112) where L-type Ca channels and SR Ca re-
lease channels co-localize (105). Thus, there is a prepon-
derance of evidence that Ca entry by means of L-type Ca
channels is the major trigger of SR Ca release and that this
trigger Ca “grades” the release of SR Ca from a single or
cluster of ryanodine receptors in close proximity. Finally,
other sources of trigger Ca including Na/Ca exchange and
voltage per se have not been shown to be sufficient quan-
February 1, 2001
titatively to account for the Ca transients recorded in
mammalian muscle. Thus, several lines of evidence point
to the SR as the primary site of origin for activator Ca in
mammalian heart muscle (113).
Interestingly, the SR seems to lose its capability to reg-
ulate diastolic Ca tightly when the cell is “Ca overloaded.”
Under these conditions, the SR releases Ca spontaneously
and asynchronously (114,115). The resultant Ca oscilla-
tions produce delayed afterdepolarizations (DADs) ob-
served when the Ca loading of the cell is excessive, as in
digitalis toxicity, and may result in lethal arrhythmias
(116).
At the molecular level, biochemical techniques and re-
cent single channel studies have provided more direct
evidence to improve our understanding of SR function
and pharmacology. The Ca release channel serves as the
pathway for the sudden systolic delivery of Ca from the
SR to the cytosol, triggered by the increment in cytosolic
Ca itself, and facilitated by the presence of ATP. This
molecule has been purified from skeletal and cardiac
muscle SR (117–119) by several laboratories using proce-
dures that exploit the high affinity of this channel to the
plant alkaloid, ryanodine. The ryanodine-binding pro-
tein has been characterized electrophysiologically as a
channel passing Ca ions and, with a several-fold lower
permeability, potassium ions. It can be blocked by the
dye, ruthenium red. Ryanodine changes the properties of
the channel in a complex manner, producing long-lasting
openings of low conductance at nanomolar ryanodine
concentrations. Thus, it causes a continuous leakage of
Ca out of the SR, which consequently loses its ability to
store and release Ca phasically in the intact cell.
The SR Ca release channel has been cloned from both
skeletal and cardiac muscle (120). The cardiac cDNA is
16,532 base pairs in length and encodes a protein of 4,969
amino acids that is 66% identical with the skeletal muscle
gene product. Four of these identical subunits are be-
lieved to assemble to form the functional Ca release chan-
nel spanning the SR membrane. The molecule is domi-
nated by a huge N-terminal domain that appears to form
the “foot processes” that span the cytosolic gap between
the SR and the transverse tubules in skeletal muscle. The
putative channel resides entirely within the carboxy ter-
minal end of the molecule. One interesting difference be-
tween the skeletal and cardiac muscle isoforms arises
from a difference in phosphorylation sites (121); the car-
diac receptor has a multifunctional Ca-calmodulin– de-
pendent protein kinase site that is absent in the skeletal
muscle isoform, hinting at possible differences in regula-
tory mechanisms (see below).
Because ryanodine acts effectively to dissociate the SR
from excitation– contraction coupling (122,123), it has
been widely used as an experimental tool (101,124,125).
A homologous mode of action has been proposed for
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 Calcium Currents and Arrhythmias/Shorofsky and Balke
high (millimolar) concentrations of caffeine, which in-
duce a contracture in heart muscle preparations owing to
a sudden release of Ca, causing Ca depletion of the SR.
The anthracycline cytostatic agent, doxorubicin, has been
proposed to affect cardiac contractility by such a mecha-
nism (126,127). In contrast to ryanodine, caffeine and
doxorubicin exert a number of additional effects. A spe-
cific antiarrhythmic drug has not yet been found to act by
interfering with SR Ca release, although this pathway has
emerged as a promising target for rational drug design
now that the specific molecular components are becom-
ing increasingly well defined.
Regulation
Despite a wealth of biophysical data under nonphysi-
ological conditions (with channels reconstituted in lipid
bilayers and conducting monovalent cations), little is cer-
tain about the physiological factors that regulate SR Ca
channel function in living cells. Presumably, Ca activates
the channel and initiates Ca-induced Ca release. Another
regulatory factor that most surely plays a prominent
pathophysiological role in ischemia is ATP; both ATP
and, to a lesser extent, Mg-ATP potentiate channel flux by
increasing open probability. In ischemia, intracellular
ATP depletion would tend to decrease Ca release by
means of the ryanodine receptors, perhaps contributing
to ischemic contractile failure (which temporally pre-
cedes the onset of electrical inexcitability).
IP3 RECEPTORS
Physiological and Pathophysiological Roles
Like ryanodine receptors, inositol triphosphate (IP3) re-
ceptors are integral SR membrane proteins that mediate
the efflux of Ca from this intracellular store. IP3 receptors
are found predominantly in smooth muscle, where they
appear to mediate drug-induced receptor-activated SR
Ca release. Although IP3 receptors are also found in the
myocardium, less is known about their functional role in
heart. In contrast to observations made in other tissues
(128,129), it appears unlikely that Ca release in cardiac SR
is primarily triggered by the second messenger IP3, al-
though a modulatory role cannot be excluded. Although
it appears that IP3 is not involved in normal cardiac exci-
tation– contraction coupling, it may have a role in the
maintenance of diastolic tension and in the physiological
modulation of contractility in response to a variety of
drugs and hormones. IP3 is produced by hydrolysis of the
membrane phospholipid phosphatidylinositol-biphos-
phate, catalyzed by the enzyme phospholipase C. A vari-
ety of agonists can activate phospholipase C in the heart
(130). In particular, the molecular basis of the positive
inotropic effect of ␣-adrenergic stimulation might in-
volve this pathway (131). Nevertheless, the inotropic ef-
136 February 1, 2001 THE AMERICAN JOURNAL OF MEDICINE威
fects of ␣-adrenergic stimulation are usually quite small
and differ among species (132,133).
Angiotensin II receptors are present on cardiac myo-
cytes, and exposure to angiotensin is known to increase
cellular IP3 levels (134). Thus, it seems plausible that the
postulated arrhythmogenic effects of angiotensin II in
heart failure may at least partly be attributable to IP3 ele-
vation, possibly as a result of a feedback effect of elevated
intracellular [Ca] on ion channels in the surface mem-
brane.
IP3 receptors are found in higher density in specialized
cardiac conducting tissue (135) and Purkinje cells (136),
consistent with a possible role in cardiac excitability, and
in intercalated disks (137), suggesting a potential role in
intercellular communication. In addition, IP3 receptors
have been implicated in cell growth, differentiation, and
apoptosis (138,139).
Structure–function Relationship
IP3 receptors are formed of four similar or identical sub-
units of 315,000 molecular weight (2,749 amino acid res-
idues) arranged in a quatrefoil pattern similar to that de-
scribed for the ryanodine receptor. Several subunits have
been cloned from cerebellar cDNA libraries (140). Hy-
dropathy analysis predicts that 80% of the peptide forms
a large cytoplasmic domain, followed by six to eight
transmembrane domains per subunit. The membrane-
spanning regions probably form the ion conductance
pathway. It is not yet clear whether the cardiac receptor
represents the same gene product, a splice variant, or an
entirely different gene from the cerebellar isoform(s).
Regulation
Little is known about the regulation of IP3 receptors aside
from the reported effects of angiotensin II and ␣-adren-
ergic stimulation. IP3 receptors are both autophosphory-
lated (141) and phosphorylated by several protein ki-
nases, including PKA (142), PKC, and Ca-calmodulin-
dependent kinase II (143). In addition, IP3 has two
potential tyrosine phosphorylation sites (144). In T lym-
phocytes, tyrosine phosphorylation of IP3 appears to
modulate intracellular Ca release and may modulate in-
tracellular Ca levels (138,145). However, this putative
role has yet to be demonstrated in cardiac cells.
CONCLUSIONS
Calcium channels are critical to normal cardiac function.
They are important in impulse generation, conduction,
maintaining the plateau phase of the action potential, and
contraction. At least three different surface membrane
channels exist and are preferentially distributed in vari-
ous areas of the heart, giving a clue as to their function.
The L-type Ca channel is the most abundant type in all
cardiac cells and is responsible for the entry of Ca into the
Volume 110
 Calcium Currents and Arrhythmias/Shorofsky and Balke
cell that triggers contraction. Inhibitors of L-type Ca
channels will depress conduction through the AV node,
thus making them useful drugs for the treatment of many
supraventricular arrhythmias. T-type Ca channels are
prevalent in the conduction system and are probably in-
volved in automaticity. Enhanced function of these chan-
nels may lead to an increase in automaticity, as is ob-
served in repetitive monomorphic ventricular tachycar-
dia and some forms of atrial tachycardias. The newly
described ICa(TTX) activates over a voltage range more
negative than the sodium channel and thus may be im-
portant in “boosting” or enhancing conduction and con-
traction. The main intracellular Ca channel resides in the
sarcoplasmic reticulum and is responsible for the ulti-
mate release of the Ca that activates the contractile appa-
ratus. Oscillatory behavior of this channel influences the
sarcolemmal membrane, causing delayed aftercontrac-
tions and arrhythmias, such as those seen in digoxin tox-
icity. The molecular characterization of each of these
channels has begun, and important structural data al-
ready has been obtained. The continued molecular deter-
mination of these channels and their regulation will en-
hance our knowledge of their normal function and dys-
function in disease states, leading to the development of
new therapeutic agents to treat arrhythmias and contrac-
tile dysfunction.
ACKNOWLEDGMENT
The authors apologize in advance to our many colleagues whose
outstanding scientific contributions to our understanding of
the general field of cardiac calcium channels could not be in-
cluded in this concise review because of the inevitable space
limitations. The authors also thank all past and present mem-
bers of their laboratories for the numerous and valuable contri-
butions to this review.
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