Transcript Presentación de PowerPoint

Ion channels are proteins that have a pore.
Channels are usually highly selective:
only specific ions can go through the
pore. Channels change its
conformation depending on various
factors. In this way they can switch
from being open to being closed. When
the channel is open, ions can flow
through the pore driven by the innermembrane electric field and the
concentration gradient.
Voltage dependent ion channels change their conformation when the
voltage across the membrane changes.
Ligand-dependent channels change their conformation when they
bind some substances (ligands).
Ion channels can be studied electrophysiologically (since
the movement of ions produces a measurable electric
current).
Voltage clamp technique
(Figures from F. Bezanilla)
Currents in response to depolarizing
pulses from -60 to 60 mV in
increment of 10 mV from a holding
potential of -70 mV.
The study of ion channels changed dramatically with the
introduction of the patch-clamp technique (Neher and
Sackman).
Patch clamp.
It allows the measurement
of currents through a
single or a few channels.
It is done with a very
thin pipette that forms a
gigaohm seal with the
membrane. There are
different configurations.
Recording currents correspond either to the opening of active ion
channels under the pipette (inside-out configuration) or to the opening
of all active ion channels present at the cell surface (whole-cell
configuration). Dashed line refers to the zero current.
From single channel experiments it is possible to obtain
the open probability of the channel (which is calculated as
the ratio between the time during which there is an
observable current and the total time of the observation).
Open Probability
Single channel records
Figures provided by D Mak and K Foskett
A canal de K+
Se pueden calcular las “dwel time distributions”
A partir de las observaciones se pueden construir modelos
cinéticos (Hidden Markov Models). Problema: muchas topologías
equivalentes entre sí!!
Nuevas técnicas de visualización y preparación permiten ver otros
procesos estocásticos que ocurren en células (que se solían estudiar en
términos de una descripción de “campo medio”).
Methods for studying ion channels
Functional
• Current clamp (record membrane potential)
• Voltage clamp (record ionic currents)
Macroscopic
2-electrode
Whole-cell patch clamp
Microscopic (single channels)
Cell-attached
Inside-out
Outside-out
• Gating current
• Spectroscopy
Structural
• Biochemistry
• Molecular biology
• Structural (x-ray, etc.)