Biomedical Engineering Reference
In-Depth Information
drug targets in the pharmaceutical industry. Drug-discovery projects today depend strongly on
large-scale blind-screening for i nding new chemical lead molecules. The only high-throughput,
high-quality technology to be used for screening on every ion channel subtype is the newly devel-
oped automated patch-clamp technique. With this method, parallel recordings are performed by a
robot on 50-100 arrays of ion channel expressing cells positioned on silicon chips. Smaller through-
puts can be obtained on arrays of 8-10 frog eggs expressing the desired ion channels, but the phar-
macology of some channels may be different in this nonmammalian system. Channels giving rise
to changes in the intracellular Ca
2+
concentration can be screened using l uorescent Ca
2+
dyes in a
384 well l uorescent reader (FLIPR) (see Chapter 12.3.2), which may also be useful for channels
causing slow voltage changes. The use of other screens is typically limited to specii c ion channels
such as rubidium or thallium l ux through K
+
channels, or ligand binding to neurotransmitter-gated
channels.
13.1.8 S
TRUCTURE
OF
V
OLTAGE
-G
ATED
I
ON
C
HANNELS
The superfamily of voltage-gated ion channels encompasses more than 140 members and is one
of the largest families of signaling proteins, following the G-protein-coupled receptors and pro-
tein kinases. The pore-forming
-subunits of voltage-gated ion channels are built upon common
structural elements and come in four variations. The simplest version is composed of two TM seg-
ments connected by a membrane-reentrant pore-loop and having N- and C-termini on the inside
(Figure 13.3). Four of such subunits form the channel. This architecture is typical for the so-called
inward-rectifying K
+
channels (K
ir
). It is found in a number of bacterial channels, suggesting it is
the ancestor of the family. The second type is made by a concatenation of two such subunits, and the
channel is formed by two double constructs. The third type is the 6-TM subunit, in which four extra
membrane-spanning N-terminal domains including a voltage-sensor have been added to the basic
2-TM pore unit. Four of these 6-TM units form a channel. The group of 6-TM channels is rather
large and includes the voltage-gated K
+
channels (K
v
), the calcium-activated K
+
channels (K
Ca
),
the cyclic nucleotide-gated (CNG) channels, the hyperpolarization-gated channels (HCN), and the
transient receptor potential (TRP) channels. Finally, the fourth channel structure type is made by
concatenating four of the 6-TM subunits, making up a 24-TM subunit that forms the channel alone.
This type is represented by the voltage-gated Na
+
and Ca
+
channels (Na
v
and Ca
v
). Within each of
the four domains the six TM segments are denoted S1-S6.
Three different parts of the channels are responsible for the functions: ion permeation, pore gat-
ing, and regulation. The narrow part of the pore is called the selectivity i lter, and this has been stud-
ied by high-resolution x-ray in crystallized K
+
channels giving valuable insight into the selectivity
mechanism (Figure 13.4). The residues in the pore loop line the selectivity i lter and their carbonyl
groups act as surrogate-water implying that the chemical energy of the dehydrated K
+
ions entering
α
2-TM
4-TM
6-TM
24-TM
N
N
N
C
C
N
C
C
K
ir
K
2P
Na
v
Ca
v
K
V
K
Ca
HCN
TRP
FIGURE 13.3
Topology of voltage-gated cation channels. (From Palle Christophersen.)
