Functional ion channels in human pulmonary artery smooth muscle cells: Voltage-dependent cation channels.

2011·2.9 MB·English

by  Firth, Amy L.

Review Article
Functional ion channels in human pulmonary
artery smooth muscle cells: Voltage-dependent
cation channels
Amy L. Firth1, Carmelle V. Remillard2, Oleksandr Platoshyn2, Ivana Fantozzi2, Eun A. Ko3, Jason X.-J. Yuan2,3
1The Salk Institute for Biological Studies, La Jolla, California, USA, 2Department of Medicine, University of California, San Diego,
La Jolla, California, USA, and 3Department of Medicine (Section of Pulmonary, Critical Care, Sleep and Allergy),
Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, Illinois, USA
ABSTRACT
The activity of voltage-gated ion channels is critical for the maintenance of cellular membrane potential and generation of action
potentials. In turn, membrane potential regulates cellular ion homeostasis, triggering the opening and closing of ion channels in
the plasma membrane and, thus, enabling ion transport across the membrane. Such transmembrane ion fl uxes are important
for excitation–contraction coupling in pulmonary artery smooth muscle cells (PASMC). Families of voltage-dependent cation
channels known to be present in PASMC include voltage-gated K+ (Kv) channels, voltage-dependent Ca2+-activated K+ (Kca)
channels, L- and T- type voltage-dependent Ca2+ channels, voltage-gated Na+ channels and voltage-gated proton channels.
When cells are dialyzed with Ca2+-free K+- solutions, depolarization elicits four components of 4-aminopyridine (4-AP)-sensitive
Kvcurrents based on the kinetics of current activation and inactivation. In cell-attached membrane patches, depolarization elicits
a wide range of single-channel K+ currents, with conductances ranging between 6 and 290 pS. Macroscopic 4-AP-sensitive Kv
currents and iberiotoxin-sensitive Kca currents are also observed. Transcripts of (a) two Na+ channel α-subunit genes (SCN5A
and SCN6A), (b) six Ca2+ channel α−subunit genes (α , α , α , α , α and α ) and many regulatory subunits (αδ, β , and
1A 1Β 1Χ 1D 1E 1G 2 1 1-4
γ), (c) 22 Kv channel α−subunit genes (Kv1.1 - Kv1.7, Kv1.10, Kv2.1, Kv3.1, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv5.1, Kv 6.1-Kv6.3,
6
Kv9.1, Kv9.3, Kv10.1 and Kv11.1) and three Kv channel β-subunit genes (Kvβ1-3) and (d) four Kca channel α−subunit genes
(Sloα1 and SK2-SK4) and four Kcachannel β-subunit genes (Kcaβ1-4) have been detected in PASMC. Tetrodotoxin-sensitive

and rapidly inactivating Na+ currents have been recorded with properties similar to those in cardiac myocytes. In the presence
of 20 mM external Ca2+, membrane depolarization from a holding potential of -100 mV elicits a rapidly inactivating T-type Ca2+
current, while depolarization from a holding potential of -70 mV elicits a slowly inactivating dihydropyridine-sensitive L-type Ca2+
current. This review will focus on describing the electrophysiological properties and molecular identities of these voltage-dependent
cation channels in PASMC and their contribution to the regulation of pulmonary vascular function and its potential role in the
pathogenesis of pulmonary vascular disease.
Key Words: Ca2+ channel, K+ channel, membrane potential, Na+ channel, pulmonary hypertension
INTRODUCTION (PASMC). Electromechanical and pharmacomechanical
coupling processes are the two major EC coupling
Intracellular ion homeostasis, cell volume and membrane mechanisms. Of these, it is the electric excitability
excitability are all important mechanisms regulated by that plays an important role in EC coupling in the
the membrane permeability to cations and anions. It is pulmonary vasculature,[1,2] predominantly controlled
this transmembrane ion flux that is the predominant
factor in controlling excitation–contraction (EC) coupling
Access this article online
mechanisms in pulmonary artery smooth muscle cells
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Website: www.pulmonarycirculation.org
Address correspondence to:
Prof. Jason X.-J. Yuan
Department of Medicine (Section of Pulmonary, Critical Care and Sleep DOI: 10.4103/2045-8932.78103
and Allergy), Institute for Personalized Respiratory Medicine,
University of Illinois at Chicago, COMRB Rm. 3131 (MC 719),
909 South Wolcott Avenue, Chicago, Illinois 60612, USA Pulm Circ 2011;1:48-71
E-mail: [email protected]
48 Pulmonary Circulation | January-March 2011 | Vol 1 | No 1
Firth, et al.: Ion channels in human PASMC
by the transmembrane ion flux in PASMC. Indeed, many define the pathogenic roles of ion channels in pulmonary
vasoactive substances also alter the membrane potential vascular disease and to develop new therapeutic
(E ) in these cells.[3,4] Expression and functionality of ion approaches for patients with pulmonary hypertension.
m
channels in the plasma membrane is also important in
modulation of cell motility, migration and proliferation As mentioned above, EC coupling requires a change in
by governing the cytoplasmic free Ca2+ concentration membrane potential to alter vascular tone. Ion channels
([Ca2+] ). are sarcolemmal pores selectively permeable to either
cyt
cations (Na+, Ca2+, K+) or anions (Cl-). Both anions and
A rise in [Ca2+] in PASMC triggers pulmonary cations are distributed on either side of the cell membrane,
cyt
vasoconstriction[5] and stimulates cell proliferation[6] and and their transmembrane movement is based on their
migration,[7] leading to pulmonary vascular remodeling.[8] electrochemical gradient, a potential- and concentration-
The mechanisms involved in the regulation of [Ca2+] directly based driving force for the ions, i.e. flowing from more-
cyt
control vasomotor tone and vascular wall thickness; two concentrated to less-concentrated zones and, for cations,
major determinants of pulmonary vascular resistance from positive or less-negative sites to those with a more
(PVR). Because PVR is inversely proportional to the negative membrane potential. In human cells, Na+ (~140
fourth power of the radius (r) of the pulmonary arterial mM) and Ca2+ (~2 mM) are the dominant cations in the
lumen (PVR=8Lη/πr4), a very small change in r would external fluid (concentrations similar to those found in
thus cause a large change in PVR. As a consequence, blood plasma), whereas K+ (~140 mM) is the dominant
pulmonary vasoconstriction will also increase PVR by cation in the cell cytoplasm. Cl-, the most dominant anion
reducing the arterial radius. Pulmonary arterial pressure in vascular smooth muscle cells,[15] is unevenly distributed
(PAP), a diagnostic criterion for PAH, is a product of PVR between the cytosol and the extracellular fluids, and plays
and cardiac output. Pulmonary vasoconstriction and an important role in controlling osmolarity, cell volume,
vascular medial hypertrophy caused by excessive PASMC excitability and ion homeostasis [Table 1]. Additionally,
proliferation and migration contribute considerably to the ion channels expressed in the plasma membrane also play
elevated PVR in patients with pulmonary hypertension. important roles in the regulation of secretion, migration,
Indeed, dysfunction of a number of ion channels has proliferation, differentiation and apoptosis. In vascular
been implicated in a variety of cardiopulmonary diseases, smooth muscle cells, the resting E is predominantly
m
such as pulmonary arterial hypertension,[8,9] spontaneous regulated by the permeability and the concentration
genetic systemic arterial hypertension[10-13] and heart gradients of K+ across the plasma membrane. The reason
failure.[14] Therefore, defining the molecular identities that the resting E (-40 to -55 mV) in vascular smooth
m
and electrophysiological properties of plasmalemmal muscle cells is not equal to the K+ equilibrium potential
ion channels in human PASMC will help to enhance our (approximately -85 mV) indicates that other cation
understanding of normal EC coupling mechanisms, to (e.g., Na+ and Ca2+) and anion (e.g., Cl-) channels also
Table 1: Ionic composition of extracellular and intracellular solutions used for measurement of various ion
channel currents
Current Na+ Ca2+ K+ Mg2+ Cl- Cs+ ATP EGTA Glu HEPES pH
Type mM mM mM mM mM mM mM mM mM mM
I (whole cell)
Na
Bath 141 - 4.7 3 151.7 - - 1 10 10 7.4
Pipette 10 - - 4 143 135 5 10 - 10 7.2
I (whole cell)
Ca
Bath 110 20 4.7 1.2 157.1 - - - 10 10 7.4
Pipette 10 - - 4 143 135 5 10 - 10 7.2
I (whole cell)
K(V)
Bath 141 - 4.7 3 151.7 - - 1 10 10 7.4
Pipette 10 0 135 4 143 - 5 10 - 10 7.2
I (whole cell)
K(Ca)
Bath 141 1.8 4.7 1.2 151.7 - - - 10 10 7.4
Pipette 10 0 (8.8) 135 4 143 - 5 - (10) - 10 7.2
I (cell attached)
K(Ca)
Bath 141 1.7 4.7 1.2 151.7 - - - 10 10 7.4
Pipette 10 - 125 4 129 - 5 0.1 - 10 7.2
I (cell attached)
K(V)
Bath 141 1.8 4.7 1.2 151.7 - - - 10 10 7.4
Pipette 5 0 137 1.2 143.2 - 5 0.1 0 10 7.2
Pulmonary Circulation | January-March 2011 | Vol 1 | No 1 49
Firth, et al.: Ion channels in human PASMC
contribute to regulating the E . This review will provide
an in-depth summary of them molecular identities and a Approach Attach Make Break into
cell to cell seal cell
electrophysiological properties of voltage-dependent Vcom
cation channels in PASMC, focusing on Na+ and Ca2+
I
channels, which are opened by membrane depolarization
and responsible for cell excitation, and voltage-gated Kv
and Kca channels, which are responsible for controlling
resting E and repolarization when the cells are stimulated.
m Cell-attached Whole-cell
Passive cell membrane properties of human b
PASMC
Cell-attached +
Na
The whole-cell patch clamp configuration[16] may be
likened to an electrical circuit [Figures 1 and 2a]. A K+ Ca2+
+
capacitor is made with two charged surfaces separated by K
a dielectric substance. The pipette itself is such a dielectric 2+ K+ K+
Ca
substance with two charged surfaces and, therefore, +
K
is represented by a capacitor in the circuit. Pipette
capacitance (C , measured in Farads, F) is complicated Whole-cell K+
p
in character, but its contribution to the overall circuit is
usually minimized electronically by injecting a current c
Open probability Activation Deactivation
transient designed to pre-charge the glass surface to the
Inactivation
new desired potential. The pore of the pipette presents
a resistance to current flow that may be easily measured
e
bwehfoolree- cseelall a fcocremssa,t hioonw (eRvee, rm, tehaiss urerseids tiann Oceh ims sin, cΩr)e. aDsuedri nbyg plitude mplitud I
further resistance to current flow due to the contents or Am A
geometry of the cell itself (“series resistance” or “access
+80 mV
resistance,” R), i.e. resistance to filling the entire cytosolic
a Open Closed Vcom
space with the desired amount of charge or potential due duration duration
-70 mV
to interaction of charges with proteins or due to limited
flux through long cell processes or narrow cell geometry. Figure 1: Patch-clamp electrophysiology. (a) Formation of the giga
seal and the subsequent cell-attached and whole-cell configurations.
Once the cytosolic space of the cell is voltage clamped, (b) Enhanced view of the membrane–pipette arrangements in the cell-
attached and whole-cell confi gurations. In a cell-attached patch, unitary
the cell membrane also presents its own capacitance.
currents are produced by ion fl ux through single channels. Three different
Unlike the pipette, the cell membrane is of relatively
channel types are shown. In the whole-cell mode, the macroscopic current
uniform thickness and uniform dielectric content recorded is the summation of all the currents generated by similar channels
(lipids); therefore, in most cells, the specific membrane throughout the cell. (c) Measured parameters. Single-channel recordings can
capacitance (C ), which is normalized by the area of the provide information relating to the amplitude the unitary currents, the open
m probability of the channels and the amount of time the channel(s) spend in
plasma membrane, is ~1 μF/cm2,[17] and a measure of
open (open duration) or closed (closed duration) confi gurations. Macroscopic
the cell capacitance is a good indicator of cell size. The
currents are characterized by the current amplitude, activation, inactivation
cell membrane itself is a very good dielectric, presenting and deactivation during a pulse protocol
a resistance of several gigaOhms (gigaΩ) when the
membrane channels are closed at rest, in effect stopping through that particular type of channel. Because Ohm’s
the flow of charge across the membrane. However, the law defines resistance as the inverse of conductance, the
membrane resistance (R ) is strongly influenced by the overall membrane resistance is the inverse of the sum
m
presence of ion conductances through the membrane ion of all the conductances present on the membrane. The
channels. simple measurement of overall membrane resistance
is therefore a good indicator of the amount of current
Ion channels are selectively permeable to specific carried through all the open channels on the membrane.
cations, and have a gating mechanism that may be As many membrane channels are voltage dependent, the
controlled by voltage or other methods. Ion channels membrane resistance likewise varies with membrane
produce a conductance (g, measured in Siemens, S) that potential.
is dependent on the transmembrane electrical potential
energy (∆E, or E , measured in Volts, V), and defined by By employing a small hyperpolarizing command voltage
m
Ohm’s law, I=gE, where I is the conductance or current step (V ), for example from -70 mV to -85 mV (close to
x x comm
50 Pulmonary Circulation | January-March 2011 | Vol 1 | No 1
Firth, et al.: Ion channels in human PASMC
the equilibrium potential for K+), current transient (I ) (capacitative) area; for PASMC, this is in the region of
tran
is induced [Figure 2b]. The cell membrane capacitance 1.25 μF/cm2, similar to the 1.3 μF/cm2 reported in rat
(C ) can then be determined by pClamp software based on caudal artery smooth muscle cells.[18] R under resting
m m
the equation: C =(integral of I )/)V ). The membrane conditions is usually very high in the vascular smooth
m tran comm
input resistance (R ) is then calculated from the equation: muscle cells.[19] Indeed, the calculated R in PASMC ranges
m m
R =(R ×R )/(R -R ), where R and R are the from 1 GΩ to 12 GΩ, with an average R of 5±1 GΩ (n=171)
m total seal seal total seal total m
resistance determined, respectively, from the steady [Figure 2d]. Importantly, the duration of PASMC in cell
currents of I in response to V (-5 mV) before and culture conditions does not significantly alter the values
tran comm
after break-in. As shown in Figure 2c, C can range from for C and R [Figure 2e and f].
m m m
15 pF to 45 pF, with an average C of 34±5 pF measured
m
in 220 PASMC. The specific membrane capacitance can Membrane potential can be measured in the current-
be calculated from the mean values of C and cell surface clamp (I=0) mode. The resting E in cultured human
m m
PASMC is approximately -45±5 mV [Figure 3a], and
is slightly less negative than that observed in freshly
a
dissociated PASMC from animals.[20,21] As previously
mentioned, E is less negative than the E (approximately
Cp
m K
Re Electrode -85 mV), which suggests that E in these cells is also
m
Cm controlled by the permeability of other ions (e.g., Na+,
Ca2+ and Cl-). The equilibrium potentials for Na+, Ca2+ and
Rm Cell
Cl- are believed to be +66, +122 and -26 mV, respectively,
in native vascular smooth muscle cells.[15] In some PASMC,
b spontaneous electrical activity has been observed under
-70 mV resting conditions [Figure 3b], suggesting that these cells
-80 mV
are electrically excitable.[22] This spontaneous electrical
Seal activity in PASMC is dependent upon the presence of
extracellular Ca2+ [Figure 3b].[2,23-25]
Break-in
200 pA
Evolution and diversity of the pore-forming
1 ms
voltage-gated cation channels
c d
70 50 Ion-selective voltage-gated cation channels generate
60 electrical activity in cells by undergoing rapid
40
er50 er conformational changes from an impermeable structure
mb40 mb30 to a highly permeable pore in the membrane through
Nu30 Nu20 which ions can pass. Based on inherent similarities
Cell 20 Cell 10
10
0 0 a
5 101520253035404550 1 234 56 7 8 9101112 30 mean Em = -45.2 mV
e Cm (pF) f Rm (GΩ)
50 8 er 20
b
7 m
40 u
6 N 10
(pF)m2300 (G)Ωm345 Cell 0-75-70 -65-60-55-50 -45-40-35-30 -25-20-15-10
C10 R 2 Resting Em (mV)
1 b
20
0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0
Time in culture (day) Time in culture (day) V)
m -20
Figure 2: Passive membrane properties of human pulmonary artery smooth (Em-40
muscle cells (PASMC). (a) The cell and pipette form a circuit in the whole-
cell patch-clamp confi guration. Membrane capacitance (C ) and resistance -60 0Ca 1 min
m
(R ) are indicators of cell size and transmembrane ion fl ux, respectively.
m Figure 3: Electrically active human pulmonary artery smooth muscle
(b) C is often used to indicate that the membrane is ruptured. In the cell-
m cells (PASMC). (a) Histogram showing the wide distribution of resting
attached confi guration (“Seal”), C , measured as the surface area under
m E in human PASMC. E was measured in the current clamp (I=0) mode.
the transient spikes, is small. Upon whole-cell access (“Break-in”), C is m m
m (b) Spontaneous action potentials recorded in human PASMC are abolished
greatly increased. (c and d) Frequency distribution of C and R within a
m m when external Ca2+ is removed. Electrical activity is restored upon return to
cell population. (e and f) C (n=220) and R (n=171) of human PASMC do
m m normal physiological Ca2+ (1.8 mM)
not vary over time in culture
Pulmonary Circulation | January-March 2011 | Vol 1 | No 1 51
Firth, et al.: Ion channels in human PASMC
in the transmembrane domain structure of Na+, Ca2+ have a common 1D-6TM cyclic-nucleotide gated (CNG)
and K+ channel pore-forming α subunits, it is widely channel ancestor, with S4 and pore regions similar to
agreed upon that voltage-gated cation channels share a voltage-gated K+, Ca2+ and Na+ channels. In addition to
common ancestor. The basic building block of all these structural similarities, CNG channels also exhibit some
channels is a one-domain (1D) two-transmembrane voltage sensitivity and are permeable to both monovalent
segment (2TM) protein with an ion-selective pore/ and divalent cations, making it an ideal common
loop region between the transmembrane segments,[26] precursor.[26] (c) More recently, Durell and Guy[29] showed
reminiscent of prokaryotic and eukaryotic K+-selective that a Ca2+ channel with a 1D-6TM motif could be detected
inward rectifier channels [Figure 4]. Indeed, K+ channels in the akylaphilic bacterium Bacillus halodurans\pard
are the oldest of the voltage-gated cation channels as plain. This suggests that more mutations conferring
examples of these have been found in both prokaryotic Ca2+ selectivity in bacterial 1D-6TM channels occurred
and eukaryotic organisms.[27] Over time, multiple gene before any gene duplication occurred and prior to the
duplications and modifications elaborated this channel development of lower eukaryotes (protozoans), where
by the addition of four transmembrane segments, forming 4D-6TM Ca2+ channels have been identified.[26,29]
1D six-transmembrane segment (6TM) protein that
constitutes the basic pore-forming α−unit of mammalian The first 4D-6TM proteins were voltage-dependent Ca2+
voltage-gated cation channels. From this point, the channels (VDCC), with early gene identification revealing
evolution of voltage-gated ion channels diverged, with ion multiple channels within the same tissue or cell. Eventually,
selectivity being a key element to channel diversification. Ca2+ channels were classified as low-voltage activated (LVA)
Figure 4 provides a simple phylogenetic tree of voltage- or high-voltage activated (HVA) VDCC (see below and
gated cation channels based on sequence identity and Catterall[30] for review). Ca2+ influx via VDCC has already
domain arrangement. been established as an effector or trigger in numerous
cellular processes, with the different channel subtypes
K+ channels evolved into the most diverse family sometimes playing different roles. The variety of functional
of channels, mainly due to the sheer number of α− roles for VDCC correlates well with the significant
subunits and possible α β subunit combinations. Four structural diversity between the 10 VDCC α subunits
4 4
superfamilies of human K+ channels have maintained the currently identified (α , α ).[30] Although the six isoforms
1A-1I 1S
1D-6TM motif, and multiple α−subunits have surfaced identified in human PASMC represent each of the five
for each: ether-a-go-go (eag, erk, elk; 3 isoforms in
human), KQT (5 isoforms in humans), Kca (maxi-Kca
and SKca; 6 isoforms in humans) and Kv (11 subfamilies
and ≥30 isoforms) (see Coetzee et al. for review[28]). The S1 S2
sequence identity varies greatly within (e.g., 35–88%
identity between Kv1 and Kv9) and between (e.g.,
8–17% identity between Kv and Kca) the families.[28]
Chromosomal site analysis of the known human isoforms
2 TM
also suggests that K+ channels have existed for a long 2 D 1 D
?
time. Genes encoding 1D-6TM K+ channels are found on KT KIR/KATP
at least 13 human chromosomes, with little evidence of
clustering of genes except in the case of a few Kv channels 6 TM
1 D 1 D
(Figure 5 depicts the chromosomal location of channel
Ca(bac) 1.1 eag Kv KQT KCa
pore-forming and regulatory subunits identified in human
PASMC). Kv channel α−subunits alone can be found on 10
Slo SK
chromosomes within the human genome. 4 D (α1) (1-4)
HVA LVA
Shaker Shaw silent
Na+ and Ca2+ channels evolved after K+ channels were CaL CaT (Kv1) (Kv3) (Kv5-6,
(α1C, D, F, S) (α1G-I) Kv8-9)
well established.[27] A few theories have been put forth
Shab Shal other
to explain the development of four-domain (4D) -6TM CaN CaR CaP/Q Na (Kv2) (Kv4) (Kv10,
channels from 1D-6TM channels:[26] (a) two rounds of gene (α1B)(α1E)(α1A)(SCN1A-11A) Kv11)
duplication of 1D-6TM K+ channels (1D÷2D, 2D÷4D) and
Figure 4: Proposed phylogenetic tree depicting the evolution of voltage-
mutations within the pore region to alter ion selectivity dependent cation channels. Pore-forming unit isoforms representing each
created the 4D-6TM Ca2+ and Na+ channels.[26] Coupled channel are shown in parentheses. (TM – Transmembrane domain; D – Domain;
with mutations within the pore to alter ion selectivity, this K – Two-pore domain K+ channel; K – Inward rectifi er K+ channel; K – ATP-
T IR ATP
evolutionary cascade would have produced the 4D-6TM sensitive K+ channel; HVA – High-voltage activated; LVA – Low-voltage activated;
Kv – Voltage-gated K+ channel; K – Long-QT K+ channel; Kca – Ca2+-activated K+
Ca2+ and Na+ channels. (b) 1D- and 4D-6TM channels QT
channel; SK – small-conductance Ca2+-activated K+ channel)
52 Pulmonary Circulation | January-March 2011 | Vol 1 | No 1
Firth, et al.: Ion channels in human PASMC
Ca2+ channel subtypes, there is only electrophysiological early metazoans. This has led to speculation that low-
evidence for the L- and T-type channels in PASMC[31,32] (also voltage activated and rapidly activating Na+ channels
see below). As for voltage-gated K+ channels, the associated evolved from Ca2+ channels in parallel with the evolution
genes are encoded on at least five human chromosomes, of the first nervous systems. Sequence analysis has
with no grouping of α−subunits encoding for similar shown that ligand-binding sites (e.g., carboxy-terminal
currents on the same chromosome. For example, α and calmodulin-binding site) may be conserved within the
1C
α , both encoding for L-type VDCC in human PASMC, are 4D-6TM voltage-gated Ca2+ and Na+ channels, suggesting a
1D
located on chromosomes 12 and 3, respectively, while similar evolutionary precursor.[33,34] Of particular interest
those encoding for T-, N-, R- and P/Q-type channels are is a putative calmodulin (CaM)-binding site located
scattered on chromosomes 17, 9, 1 and 19, respectively in the carboxy-terminal regions of both Na+ and Ca2+
[Figure 5]. Furthermore, while the structure of T-type channels.[33,34] Cloning of the first LVA channels verified
VDCC is very similar to that of LVA channels, the sequence that voltage-dependent Na+ channels did evolve from
identity between them is <25%, implying that the HVA and T-type VDCC.[35] Eleven known Na+ channel α−subunit
LVA subfamilies represent radically different evolutionary genes (SCN1A-11A) bearing strong biophysical and
branches.[26] sequence (~75% sequence identity) similarities have
been identified in the skeletal, cardiac and uterine muscles
Like T-type Ca2+ currents (I ), Na+ currents are rapidly and in the human brain,[26,36,37] and bear approximately
Ca(T)
activating transient currents activated at more negative 75% sequence identity to each other.[26] Because the
membrane potentials. In lower eukaryotes, Ca2+ was the isoforms bear strong similarities, even when expressed
primary charge carrier;[26] purely Na+-dependent action in heterologous systems, voltage-gated Na+ channels
potentials were not common until the advent of the are not generally grouped into families like their Ca2+
1
Chromosomal Location of Ion Channel Genes Expressed
in Human Pulmonary Artery Smooth Muscle Cells
Kvβ-2
2
3
Kv5.1
Kv9.3
5 7 11
Kv10.1
Kv3.4 (Kv6.3)* SCN11A 9 10 Kv3.1
Kv1.3 12
Kv1.10 Kv11.1 VDCC-α1C
Kv1.2 VDCC- VDCC- Kv1.5
α1D Kv1.6
β2
Kv1.1
SCN8A
VDCC-β3
K -SK3 SCN7A VDCC- MaxiK- Kv1.4
Ca
VDCCβ4 α2δ1 α1
VDCC- SCN2A
α1E SSCCNN39AA K -SK2 Kv4.2 SCN2B MaxiK-β4
Ca
VDCC-
Kvβ-1 α1B
MaxiK-β2
MaxiK-β3
MaxiK-β1
X
16
17 19 20
Kvβ-3 18 VDCC- Kv4.1
α1A
50 million
nucleotide 1μm SCN1B
K -SK4
pairs VDCC-β1 KCva1.7 Kv9.1
Kv6.1
VDCC-α1G Kv3.3
SCN4A VDCC-γ6 Kv2.1
Kv6.2
Kv6.3
Figure 5: Chromosomal location of ion channel genes expressed in human pulmonary artery smooth muscle cells (PASMC). All isoforms of pore-forming and
regulatory subunits of Na+, voltage-dependent Ca2+ channels, Kv, and Kca channels identifi ed in human PASMC are shown. Chromosomal location is based
on the primer sequences described in Table 2
Pulmonary Circulation | January-March 2011 | Vol 1 | No 1 53
Firth, et al.: Ion channels in human PASMC
and K+ counterparts. The SCN5A and 6A isoforms currents determined by the overlap between the activation
expressed in PASMC are typically found in cardiac and and inactivation curves are in the voltage range of -60
uterine muscle. Unlike K+ and Ca2+ channel α−subunit to -20 mV in human PASMC cultured in growth medium
genes, all Na+ channel α−subunit genes map within four [Figure 6b, right panel]; these values are similar to those
chromosomes (2, 3, 12 and 17) containing homeobox in other vascular smooth muscle cells, suggesting the
(HOX) gene clusters.[37] HOX genes have been predicted participation of Na+ currents in the regulation of resting
to have existed in ancestral chordates, suggesting that E in human PASMC.
m
the initial expansion of Na+ channels is associated with
multiple chromosome duplications occurring after the a
I(%)
TP
divergence from invertebrate to pre-vertebrate chordates. 100
+80 mV
The fact that many of the SCN genes are clustered on 50 pA
I(%) +60 mV 10 ms
two chromosomes also suggests that intrachromosomal 100 0
duplications also occurred over time. 23 45ms +40 mV
+20 mV
0
Voltage-gated Na+ Channels 8 16 24ms 0 mV
In a variety of excitable cells, including smooth muscle -20 mV V (mV)
cells, voltage-gated Na+ channels are responsible for -80-40 80 40
-40 mV
generating action potentials. Activation of the channels
-60 mV
induces membrane depolarization and thus increases
-80 mV
[Ca2+] by promoting Ca2+ influx through the sarcolemmal
cyt -80 mV
VDCC and the reverse mode Na+/Ca2+ exchanger.[38,39]
While the activation of Na+ channels may underlie the -70 mV -80 mV -5I 0(p0A)
spontaneous action potentials observed in cardiac and
b
skeletal muscle myocytes,[40] removal of extracellular 100 100%)
[oFCFfua i2rPg+tA uhaSrbeeMro mC3li sboih]sr,e eidsn,u dvsgupogcoletensadttg aibenny-geg omatuhtuesal dtta i cptNthlieaeo + ni eoc lpnheo cacttnhernanicnetainlalse l selm xficunainy tPa cAstbieSiolrMnivtsCye. -20 mV 0 mV55 0m spA I/I (%)max257505 257505 (/g, maxNaNa
g
0 0
as a pathway for Ca2+ entry under physiological and -70 mV
-120 mV -120 -80 -40 0
pathophysiological conditions.[41,42] V (mV)
c
Biophysical properties of voltage-gated Na+ Cont NMG Wash
a
currents (I )
Na 50 pA
The inward I observed in PASMC possesses similar 5 ms
Na(V)
biophysical and pharmacological characteristics to those
previously identified in other human vascular smooth Cont TTX Wash
b
muscle cells:[39,43-46] sensitivity to tetrodotoxin (≤1 μM
50 pA
for total inhibition), -60 to -50 mV activation threshold, 5 ms
-15 to 10 mV peak amplitude potential, -70 to -65 mV 0 mV
half-inactivation voltage (τ ≤4 ms) and -25 to -15 mV -70 mV
inact
half-activation voltage (τ ~1 ms). In cultured human
act
PASMC dialyzed with Cs+-containing solution, a rapidly Figure 6: Electrophysiological and pharmacological properties of voltage-
gated Na+ currents (I ) in human pulmonary artery smooth muscle cells
inactivating inward Na+ current is observed in the absence Na(V)
(PASMC). Cells are dialyzed with a Cs+-containing pipette solution [Table
of extracellular Ca2+ [Figure 6a]. As mentioned above, the
1]. (a) Representative currents were elicited by depolarizing the cell to
current activates at potentials close to -60 mV and peaks from a holding potential of –70 mV to test potentials between –80 mV and
at approximately +10 mV [Figure 6a]. These currents also +80 mV (protocol at bottom). Upper left inset: Steady-state activation and
inactivate rapidly, with the half-inactivation (V ) occurring inactivation of currents occurred within <5 ms and <16 ms, respectively.
0.5 Lower right inset: Summarized I I-V relationship. (b) Currents were
at approximately -65 mV and complete inactivation occurs Na(V)
evoked by a step depolarization to 0 mV from different conditioning potentials
at -20 mV [Figure 6b]. Equimolar replacement of external
(-120 mV and -20 mV) applied for 10 s prior to the test depolarization
Na+ with N-methyl-D-glucamine (NMDG) or extracellular (left). Voltage-dependent steady-state availability (I/I ) and normalized
max
application of 1 μM tetrodotoxin (TTX) is sufficient to conductance-voltage relationship (g /g ) of the peak I amplitude. The
Na Na, max Na(V)
abolish the currents [Figure 6c], suggesting that the I/Imax and gNa/gNa, max curves were best fi tted using exponential and Boltzman
equations, respectively. (c) I is completely suppressed by equimolar
currents in PASMC are carried by Na+ influx through the Na(V)
replacement of extracellular Na+ with N-methyl-D-glucamine (NMG) or
TTX-sensitive, voltage-gated Na+ channels similar to those
extracellular application of 1 μM tetrodotoxin. Currents were elicited by
described in neurons and cardiomyocytes.[40] The window step depolarizations from –70 mV to 0 mV
54 Pulmonary Circulation | January-March 2011 | Vol 1 | No 1
Firth, et al.: Ion channels in human PASMC
Voltage-gated Na+ channel genes expressed in not yet been detected in PASMC. All of these isoforms
human PASMC are expressed in the brain [Figure 7b]. The combined
A complex of three glycoprotein subunits form functional functional and molecular identification of Na+ currents
Na+ channels: a pore-forming α−subunit and two and channels in human PASMC suggest that voltage-
β-subunits that modulate channel gating and membrane gated Na+ channel activity and expression may relate
expression [Figure 7a].[47] The α−subunit alone can form a to PASMC excitability, contractility, proliferation and
functional channel and is composed of four domains, each differentiation.
containing six transmembrane segments (S1–S6) and a
pore loop (P region). Each S4 segment is believed to act Phenotypical change of voltage-gated Na+ channel
as a voltage sensor, while the S5-pore loop-S6 segments expression in freshly dissociated and cultured VSMC
form the transmembrane pore itself. Using reverse Voltage-gated TTX-sensitive Na+ currents (I ) have been
Na(V)
transcriptase-polymerase chain reaction (RT-PCR), described in several types of human vascular smooth
seven Na+ channel-related gene transcripts (SCN1B, 2A, muscle cells cultured from the aorta[43,44] and coronary[39,46]
2B, 4A, 8A, 9A and 11A) have been detected in PASMC and pulmonary[43,45] arteries. On only rare occasions have
[Figure 7b]. Transcripts for SCN5A and SCN6A have I been recorded in freshly dissociated human vascular
Na(V)
a Voltage-dependent Na + Channel
β subunit α subunit β subunit
COOH COOH
+ + + +
β1 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 β2
+ + + +
NH2 NH2
NH2 COOH
b
SCN SCN SCN
bp M 1A 1B 2A 2B bp M 3A 3B bp M 4A 5A 6A
Br 300 Br Br
200 300
300 PA 300 PA 300 PA
SCN
bp M 7A 8A 9A 10A 11A
300 Br M GAPDH
650
300 PA
c SCN1A
SCN3A
SCN2A
SCN9A
SCN8A
SCN4A
SCN5A
SCN6A
SCN7A
SCN2B
SCN1B
0.1 SCN3B
SCN10A
SCN11A
Figure 7: Molecular identity of voltage-gated Na+ channels in human pulmonary artery smooth muscle cells (PASMC). (a) Structural arrangement of Na+ channel
α-, β- and β-subunits. (b) The mRNA expression of cloned Na+ channels in human brain (Br) and PASMC (PA). Polymerase chain reaction (PCR)-amplifi ed
1 2
products displayed for the transcripts of SCN2A, SCN4A, SCN5A, SCN6A and β-actin. “-RT”, PCR performed with no reverse transcriptase (RT). “M”, 100
bp DNA ladder. (c) A phylogenetic tree showing the inferred evolutionary relationships among different Na+ channel genes
Pulmonary Circulation | January-March 2011 | Vol 1 | No 1 55
Firth, et al.: Ion channels in human PASMC
smooth muscle cells, although they are readily detected cardiomyocytes: L-type, T-type, P/Q-type, R-type and
when the same cells are cultured.[46] Although it may be N-type.[54,55] These Ca2+ channels have been sorted based
due to a technical problem (e.g., the rapid inactivation of on their electrophysiological, pharmacological, kinetic and
the currents and the large size of most freshly dissociated molecular properties. VDCC have also been separated into
smooth muscle cells to record I ), the relative inability two groups based on their activation voltage. HVA VDCC
Na(V)
to detect I in freshly dissociated, but not cultured, cells include all but T-type channels, with the latter classified as
Na(V)
from the same vascular bed may bear some relation to cell LVA VDCC. HVA channels activate at membrane potentials
dedifferentiation and proliferation.[46] More specifically, between -50 mV and -20 mV, while LVA channels activate at
voltage-gated Na+ channel expression and activity may more negative potentials approximating -70 mV. Typically,
be required to facilitate the transition from a “contractile” only currents generated by L- and T-type channels have
to “synthetic” or “proliferative” phenotype.[48,49] However, been measured in cardiovascular tissues, while all current
I have been recorded in both freshly dispersed rabbit[24] types have been recorded in neuronal tissues.[54]
Na(V)
and cultured human[45] PASMC. This raises the possibility
that the development and expression of functional voltage- Whole-cell VDCC currents (I )
Ca
gated Na+ channels in cultured cells acts as a trigger for cell In human PASMC, a large, slowly inactivating inward
differentiation and proliferation, possibly via enhanced Ca2+ current is observed when cells are held at -70
[Ca2+] , as discussed below. mV and depolarized to 0 mV [Figure 8a]. The current
cyt
activates close to -20 mV, with a maximal activation of
Functional properties of voltage-gated Na+ approximately +15 mV. Removal of extracellular Ca2+
channels in human PASMC abolishes the currents, confirming that the currents are
Na+ channels appear to play an important role in the due to Ca2+ influx. Nifedipine, a dihydropyridine blocker
regulation of [Ca2+] and sarcolemmal Ca2+ influx by of VDCC, is also able to significantly inhibit the currents.
cyt
different mechanisms. Firstly, in cardiac myocytes, The currents present in PASMC are, therefore, mainly due
enhanced TTX-sensitive I causes a localized transient to Ca2+ influx through dihydropyridine-sensitive L-type
Na(V)
increase in [Na+] , thereby activating reverse-mode Ca2+ channels. Less frequently, and while being held at a
cyt
Na+/Ca2+ exchange and increasing [Ca2+] with the very negative potential (-100 mV), depolarization to a test
cyt
subsarcolemmal space between the plasma membrane potential to -20 mV can elicit a rapidly activating transient
and SR.[41] The Ca2+ newly introduced into the cytoplasm inward Ca2+ current [Figure 8b and c]. This transient
can then trigger further Ca2+ release from the SR (which
ultimately will cause contraction and stimulate proliferation
a b
and migration) or replenish SR Ca2+ pools by Ca2+-ATPase-
0 mv -20 mv
mediated re-uptake.[42,50] Secondly, TTX-sensitive Na+
-70 mv
channels are promiscuous, i.e. they can allow permeation -100 mv
of other cations (such as Ca2+) under certain conditions
(e.g., absence of extracellular Na+, presence of tracing 100 pA
doses of steroids such as ouabain and digoxin).[42,51] 50 ms
Ca2+ influx through promiscuous Na+ channels can
contribute to local and global cardiac Ca2+ signaling, L-Type T-Type
especially in heart failure patients treated with digoxin.[52]
In addition to its modulating [Ca2+] , the permeability of c
cyt 100 100
Na+ channels to Ca2+ may also play a role in the contractile- T
to-proliferative cellular transition. Thirdly, voltage-gated %) 80 L %) 80
Na+ channels are essential in the generation of action (x60 (x60
a a
m m
p[Coate2+n]tia lbs aisne dm aonny eevxicditeanbclee cfreollms, tehxeprreebsys eredg SuClaNti5nAg I/I 2400 / II 2400 L
cyt
channels,[33,53] we can speculate that Ca2+/CaM-mediated 0 0 T
regulation of voltage-gated Na+ channels may play an 0 5 10 15 20 25 0 80 160 240
important role in the coupling of human PASMC excitation Time (ms) Time (ms)
and contraction.
Figure 8: Electrophysiological and pharmacological properties of L- and
T-type voltage-dependent Ca2+ currents (I ) in human pulmonary artery
Ca
VDCC smooth muscle cells (PASMC). Cells were dialyzed with a Cs+-containing
In excitable cells, the opening of VDCC is a critical pipette solution [Table 1]. (a) A representative current (L-type ICa), elicited
by depolarizing a cell from a holding potential of -70 mV to 0 mV. (b) A
mechanism responsible for muscle contraction induced
representative current (L-type I ), elicited by depolarizing a cell from a
by neuronal and humoral stimulation. There are at Ca
holding potential of -100 mV to -20 mV. (c) Activation (left) and inactivation
least five types of VDCC described in neurons and (right) kinetics of L-type and T-type I
Ca
56 Pulmonary Circulation | January-March 2011 | Vol 1 | No 1
Firth, et al.: Ion channels in human PASMC
current activates and inactivates rapidly in comparison demonstrated in vascular smooth muscle cells.[19,65,66] This
with the L-type current [Figure 8a], with a threshold review focuses only on the voltage-dependent channels;
potential for activation of approximately -36 mV at a Kv and Kca channels.
holding potential of -90 mV. The biophysical properties of
these currents are very similar to the T-type Ca2+ current Classification based on unitary conductance
observed in aortic[56] and renal artery[57] smooth muscle Macroscopic currents of Kv (I ) and Kca (I ) channels
K(V) K(Ca)
cells, rat PASMC[58] and cardiomyocytes.[59] can be readily dissociated based on their pharmacological
properties and Ca2+-dependence. Additionally, the single-
Endogenously expressed genes that encode VDCC channel conductance for each of these channels can also
in human PASMC serve to distinguish them from each other. The traces
As for voltage-gated Na+ channels, the pore-forming shown in Figure 10 are representative cell-attached
VDCC α -subunits (10 identified isoforms) are recordings from PASMC, where multiple channel subtype
1
composed of four, six transmembrane segment domains openings can be recorded from the same patch using
[Figure 9a] that, when expressed alone, can create identical Ca2+-containing perfusion solutions. As shown
functional channels.[47] The pore-forming S5-loop-S6 in Figure 10a, large amplitude K+ currents (a) and several
segments and the voltage-sensing S4 segments are small amplitude currents (b–f) can be recorded in a cell-
integral to the function of α -subunits. Three different attached membrane patch. In addition to the various
1
regulatory subunits are also part of the greater Ca2+ amplitudes of the recorded K+ currents, the duration of
channel complex.[54,60-62] β−subunits (four isoforms the channel openings varies in human PASMC. Examples
with their associated subtypes) play multiple roles in of long-lasting channel and “flickery” openings are
regulating channel membrane expression of α -subunits, shown in Figures 10b and c. In cell-attached patches
1
current kinetics and biophysical properties. Extracellular of PASMC, multiple amplitudes of outward K+ currents
α δ-subunits (two isoforms plus their subtypes) that can be elicited by steadily holding the patch at different
2
are attached to the plasma membrane via a disulfide potentials. Representative openings for channels
linkage can influence current amplitude and inactivation with seven different conductance levels are shown in
rates, and likely play a major role in stabilizing the Figure 11. The large amplitude current (225 pS and 189
incorporation of the Ca2+ channel complex into the plasma pS) openings are likely generated by the activation of
membrane. Finally, γ-subunits (six known isoforms) may large-conductance Kca channels,[67] while the 33 pS, 81
modulate channel assembly and channel subtype-specific pS and 6 pS channels may represent unitary currents
current kinetics, both effects being highly dependent through different Kv channels or small to intermediate
on the nature of the co-expressed β− and α δ-subunits conductance Kca channels.
2
[Figure 9a]. At the RNA level, transcripts for six pore-
forming α -subunits have been detected in human In addition to its regulation of current amplitude,
1
PASMC, encoding for all five VDCC types: α (P/Q-type), membrane potential can also affect the gating properties
1A
α (N-type), α and α (L-type), α (R-type) and α of these channels, e.g. the open probability (P ). For the
1B 1C 1D 1E 1G open
(T-type) [Figure 9b]. Additionally, a variety of regulatory 189 pS channel shown in Figure 10, P increased with
open
subunit isoforms are also present, including α δ membrane depolarization from 0.0005 at 0 mV to 0.014
2 1
[Figure 9c], β [Figure 9d] and γ [Figure 9e] in at +50 mV and 0.27 at +90 mV. Similarly P for the 33
1-4 6 open
human PASMC. From the current molecular and pS channel increased from 0.04 at +60 mV to 0.43 at +90
electrophysiological evidence, it may be speculated that mV, from 0.009 at +40 mV to 0.01 at +90 mV for the 141
the α -subunit may encode the L-type VDCC while the α pS channel and from 0.007 at +40 mV to 0.02 at +90 mV
1C 1G
encodes for the T-type VDCC in human PASMC. for the 6 pS channel. Therefore, both the single channel
amplitude and the open probability of Kca and Kv channels
Voltage-gated K+ Channels are influenced by membrane potential in human PASMC.
Functionally, both voltage-gated (Kv) channels and Ca2+-
activated K+ (Kca) channels (see below) are sensitive Whole-cell voltage-gated K+ (Kv) currents
to voltage changes. In other words, these channels are In order to record optimal whole-cell (macroscopic)
activated by membrane depolarization and are deactivated Kv currents (I ), cells are commonly perfused with
K(V)
by membrane hyperpolarization. A fundamental difference Ca2+-free bath solution (plus 1 mM EGTA) and dialyzed
between Kv and Kca channels is their response to Ca2+: in with Ca2+-free pipette solution (plus 10 mM EGTA).
vascular smooth muscle cells, Kv channels are inhibited Depolarizing the cells from a holding potential of -70
by cytoplasmic Ca2+[21,63] and Kca channels are activated mV to a series of test potentials ranging from -60 mV
by cytosolic Ca2+.[19,64] The existence of other types of K+ to +80 mV elicits outward K+ currents, with a threshold
channels, such as inward rectifier (K ), ATP-sensitive potential of activation at approximately -45 mV. Four
IR
(K ) and tandem-pore (K ) channels, has also been families of whole-cell I currents can be distinguished
ATP T K(V)
Pulmonary Circulation | January-March 2011 | Vol 1 | No 1 57