Presentation on theme: "Ion channels are proteins that have a pore. Voltage dependent ion channels change their conformation when the voltage across the membrane changes. Ligand-dependent."— Presentation transcript:
Ion channels are proteins that have a pore. 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). 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 inner- membrane electric field and the concentration gradient.
Ion channels can be studied electrophysiologically (since the movement of ions produces a measurable electric current). Voltage clamp technique Currents in response to depolarizing pulses from -60 to 60 mV in increment of 10 mV from a holding potential of -70 mV. (Figures from F. Bezanilla)
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. The study of ion channels changed dramatically with the introduction of the patch-clamp technique (Neher and Sackman). 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). Figures provided by D Mak and K Foskett Single channel records Open Probability 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.)