Transcript A blocking particle swings into and out of the channel mouth

Light-activated ion channels
for remote control of neuronal
firing
Banghart, M. et al
Choi, Yu Yong
Background
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Techniques for controlling neural activity have considerable limitations
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Traditional electrical and neural chemical methods requires invasive electrodes and
chemical delivery systems that cannot control patterns of activity in densely packed
neural tissue
Optical techniques utilizing caged neurotransmitters are less invasive and more precise,
but reversal of the effects of the uncaged transmitter is limited by its diffusion kinetics
Exogenous expression of genes encoding ion channels has been used to influence
electrical activity in specific neurons, but the onset and reversal of gene expression is
slow
Photic regulation has been conferred on neurons by introducing a rhodopsin-based
signal transduction-based cascade, This technique requires coordinated exogenous
expression of three different genes and produces light responses that can be slow in
onset and offset and variable in different neurons, possibly because the nature of the
native ion channels that are regulated by the cascade can vary between neurons
This light-activated ion channels allow rapid, remote, and noninvasive control.
Because the light-activated gate is covalently linked to the ion channel, and
because the ion-channel is integral to the neuronal cell membrane, control over
individual neurons is spatially accurate and does not rely on a diffusible ligand.
In addition, the gate can be reversly photo-switched, allowing recurrent control
of neural activity
Rod cell
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Figure 11.7. Details of phototransduction in rod photoreceptors. (A) The molecular structure of rhodopsin,
the pigment in rods. (B) The second messenger cascade of phototransduction. Light stimulation of
rhodopsin in the receptor disks leads to the activation of a G-protein (transducin), which in turn activates a
phosphodiesterase (PDE). The phosphodiesterase hydrolyzes cGMP, reducing its concentration in the outer
segment and leading to the closure of sodium channels in the outer segment membrane
Action potential
ion channels
• Three physical models for the opening and
closing of ion channels
– A localized conformational change occurs in one
region of the channel
– A generalized structural change occurs along the
length of the channel
– A blocking particle swings into and out of the
channel mouth
• Types of
– Ligand-gated
– Voltage-gated
– Stretch or pressure-gated
Shaker K+ channel
•
Typical voltage-gated K+
channels is an assembly
of four identical (or similar)
transmembrane subunits
surrounding a central pore.
Each subunit has six
transmembrane (S1-S6),
with both N- and Ctermini on the intracellular
side of the membrane
(top left panel). The
narrowest part of the pore,
the selectivity filter, is
formed by a loop between
S5 and S6; the voltage
sensor includes the S4
region with its multiple
positive charges (Yellen,
G., 2002. Nature )
Shaker K+ channel
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Liu, Y. et al. 1997. Neuron
Shaker K+ channel
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Blaustein, R. et al. 2000. Nat. Struct. Biol.
Patch Clamp
• http://www.iacusnc.org/Methods/wholecell/equipment
.html
Planar patch clamp
Xenopus laevis
Patch Clamp
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Note that only the oncell patch and the
whole-cell patch are
associated with the
complete neuron. The
others are only
concerned with a
portion of membrane.
Only the whole-cell
approach actually
accesses one of the
plasmas within the
neuron directly. In the
case of the on-cell
approach, the
impedance of the
membrane must be
considered in the
overall test set design.
Planar patch clamp
Photocontrol of MAL-AZO-QA-modified
Shaker channels in X. laevis oocytes
Discussion
• By combining synthetic chemistry and protein
mutagenesis, the Shaker channel has been
reengineered
• This technique lies in its spatial and temporal
accuracy, its noninvasiveness and its reversibility.
• and may provide an effective means for controlling
the activity of specific neurons downstream from
sites of neural damage or degeneration
• and may be used to restore light-regulated activity in
healthy retinal neurons after degeneration of rods and
cones, the native phtoreceptors
• and may have application in fields of nanotechnology,
bioelectronics and material sciences