Danusa Menegaz, Ph.D

CCB/BQM/UFSC

Patch-Clamp:Bioelectricity as a therapeutic target

Universidade Federal de Santa CatarinaDepartamento de Bioquímica e Eletrofisiologia

NUBIOCEL (Núcleo de Bioeletricidade Celular)

Patch-ClampVoltage-clamp (mV) & Current-clamp (pA)

Allows the investigation of an unique channel

or multiple channels in individual cells;

Allows voltage-clamp to study currents (pA)

and current-clamp to study voltage (mV);

The principle of the method it is to isolate a

piece of the membrane electrically from the EM

and to register the ion flow through channels

with a high conductance electrodo;

Ion channels are involved in different

physiological functions like neurons signaling,

muscle contraction, cardiac rhythm, hormone

secretion and cell volume;

1660 (Jam Swammerdam) electrical stimuli with with silver wire observed muscular contraction in frogs;

1790 (Luigi Galvani) discovered ion channels and observed the nerve impulse between motor neuron and muscle in frogs;

1850 (Hermann Helmholtz) velocity of propagation of a nerve impulse it was in miliseconds;

1952 (Hodkin; Huxley), first register with intracelular electrodo – action potential in giant axon of squid 0.5 mm; nobel prize in 1963;

1976 (Neher; Sakmann), patch-clamp discovered, single-channel, nobel prize in 1991;

History from eletrophysiology to Patch-Clamp

Patch-Clamp Set-Up

Isolated cellsTissue slices

Diseases involving ion channel function

Action potential in excitable cells

Pathological changes in the frequency of electrical

action potentials in neurons

Kv

Electrical activity of pancreatic β-cell

Electricity couple secretion in

Sertoli cells

Plasma membrane transport

Types of ion channels and activation factors

Electrochemical Gradient

Physiological solutions for the experiments

Extracellular media Intracellular media

Osmolarity of the IC and EC

OSMOMETER

Equipments for the patch-clamp technique

Plataform:

Anti-vibration tables;

Mechanics and Optics:

Microscope;

Micromanipulators;

Pipettes;

Perfusion System;

Eletronics:

Faraday cage;

Amplificators;

Digitalizer and data acquisition;

Noise;

Patch-Clamp Set-Up

Isolated cellsTissue slices

Electrical circuit

Microelectrodo – Pipette Puller

Steps to reach cell-attached configuration for the single-channel measurements

Patch-Clamp technique configurations

Single-channel

configurations

Whole-cell configurations

Current pulse (current-clamp) for the

voltage measurements (action potential)

Voltage pulses (Voltage-clamp) for current measurements

K+ and Cl- Channels

Na+ and Ca++ Channels

Sodium and Potassium channels – experimental solutions to avoid

other currents

K+ Channel Na+ Channel

Kv

Electrical activity of pancreatic β-cell

Kv channels in rat pancreatic islet

Chloride channels in Sertoli cells

Configuração Whole-cell (current-clamp)

Eletrophysiology and Molecular Biology

Thank you very much