From the Voltage Clamp to the Patch-Clamp

By Tyler Shewbert

Abstract

Alan L. Hodgkin and Andrew F. Huxley wrote a series of five papers in 1952 in which they developed an electrical model for the action potential within the membrane of the squid axon. This model was the first quantitative model describing the electrical workings in nerve cells [1]. The experimental technique that they used was the voltage clamp method, which was improved by Hodgkin by eliminating the differences in membrane potential, allowing for the measurement of the ion current flowing in and out of the cell [1, 2]. The success of the H-H model led to the development of the patch-clamp method by Bert Sakmann and Erwin Neher in the 1970s [1]. The patch-clamp method has revolutionized the study of ionic current within cell membranes because it allows accurate measurement to be taken of small, excitable and nonexcitable cells, and the ability to measure the currents within single ion channels. However, Sakmann and Neher’s success was built upon the success of the H-H model and the voltage clamp method showing the importance of research that lays the foundation for major breakthroughs. [1, 3, 4].

Introduction

The voltage clamp method is thought to have been first used by Kenneth Cole and George Marmots of Wood Hole as a method for measuring squid axons [1]. However, the breakthrough use of the voltage clamp was developed by Hodgkins and Huxley. In previous experiments there had been an issue of electrode polarization which they overcame by using two electrodes, creating the same potential across the squid membrane.  Hodgkins and Huxley then could accurately measure the ionic currents flowing in and out of the membrane [1, 2]. This enabled Hodgkins and Huxley to develop a mathematical model for current flow through the membrane. This model became the basis for future electrophysiological research.

The voltage clamp method did not allow for the measurement of individual ionic current channels within the membrane or smaller sized cells.  The patch-clamp method that Bert Sakmann and Erwin Neher developed in the 1970s allowed for the measurement of individual ionic current channels, even in small cells, including mammalian cells [1, 3, 4]. The patch-clamp technique has been improved since the 1970s allowing researchers to improve the accuracy of their current measurements and examine single channels within most cell types [3, 4]. This technique has been a boon to electrophysiological researchers ever since.

Results and Discussion

            The key to Hodgkins and Huxley success in the 1952 papers was the adjustments they made to the voltage clamp method that enabled the membrane of the squid axon to be kept at the same potential so that accurate measurements of the current flowing through the membrane could be recorded [1, 2]. There were limitations to the voltage clamp technique. The individual ion channels flowing in and out of the membrane could not be measured [1, 5]. The accuracy was effected by signal noise[1]. The method could only be used on nerve cells large enough to attach the pipettes necessary for current measurement to, hence the use of the squid axon [1, 5]. Even with these limitations, Hodgkins and Huxley developed their mathematical model of action potential through nerve membranes with remarkable accuracy that still serves as a basis for modern studies.

Bert Sakmann and Erwin Neher began developing the patch-clamp method in the 1970s [5]. This technique revolutionized the study of the action potential and ionic current channels. The main contributions of the patch-clamp method was its ability to reduce the signal to noise ratio of the measurement, the ability to take measurement of currents flowing through single ionic channels, and the ability to measure the ionic channels of smaller cells, including mammals [1, 3, 4].

The patch-clamp method has its roots in the voltage clamp method used by Hodgkins and Huxley. Instead of using two electrodes to overcome the polarization of the membrane, Sakmann and Neher used small, heat polished pipettes with electrodes the size of 0.5-1.0 mm which were filled with a saline solution and electrically sealed to the membrane of the cell through the application of a slight suction to the pipette [4]. Sakmann and Neher had transistors available to improve the amplification of the measured current while Hodgkins and Huxley only had vacuum tubes available to them [4]. Sakmann and Neher found that by using this technique they could achieve an electrical seal around 50 MW which allowed high resolution current measurements of single ion channels [3, 4]. However, Sakmann and Neher found that while this enabled accurate measurements of the ion channels within mammalian and other smaller cells to be performed, there was noise from the saline bath and pipette, and the current from the pipette and membrane was different [3-5]. A basic overview of the patch-clamp method can be seen in Figure 1.

Figure 1: An overview of the basic concept of the patch-clamp technique (from [5]).

In a 1981 paper Hamill, Neher, Sakmann, and Sigworth presented an “improved patch-clamp technique” [3]. In this paper, the authors described an improved method that would allow the electrical seal between the pipette and membrane to achieve resistances of in the gigaohm range [3]. This was accomplished by taking extra precautions to make sure the pipette surface was kept clean and suction was applied to the pipette interior  [3]. As the resistance of the electrical seal is increased, the noise is reduced allowing for improved resolution in the recording of the current [3-5]. They reported that they were able to get gigaohm seals almost all of the cell types they tried [3].

This order of magnitude improvement from the original technique has had profound impacts on the study of electrophysiology. The patch-clamp method has enabled researchers in neuroscience to examine the ion channels within nerve cells [5]. In the past twenty years, the patch-clamp method has been used in a “variety of excitable and nonexcitable cell types, ranging from neurons to lymphocytes”, therefore expanding its use outside of the realm of neuroscience [6].

Since Hodgkins and Huxley first measured the action potential in the squid axon, their mathematical model has held. This was revolutionary since it finally proved the hypothesis that Galvani had proposed 150 years before that there was some sort of electricity within animals. Once Hodgkins and Huxley had developed a mathematical foundation other methods could be developed such as the patch-clamp. Hodgkins and Huxley did the best they could with the resources they had. The current measured from the membrane needed to be amplified, but the transistor was not yet in common use, so they were working with vacuum tubes [1]. For Sakmann and Neher, the understanding of the voltage clamp method and the H-H model coupled with the advances in amplification technology allowed them to break through the restrictions that Hodgkins and Huxley faced. By developing the patch-clamp method, Sakmann and Neher opened electrophysiology to new cells types of all sizes, with improved resolution [5]. Hodgkins and Huxley laid the groundwork for Sakmann and Neher’s breakthrough which has contributed to the electrophysiology research in the last forty years.

 

Outlook and relevance of work

            The research performed by both teams won the Nobel Prize in Physiology or Medicine: Hodkgins and Huxley in 1963, and Sakmann and Neher in 1991 [1]. This recognition is well deserved. The H-H model has stood up to testing in the past six decades since its origination [1]. Hodkgins and Huxley finally formalized ideas that had been put forth by Galvani 150 years before. Their improvement of the voltage clamp method was essential in the development of the field of electrophysiology. If they had not been able to create an isopotential membrane, their experiments would not have been successful [1, 2, 6]. The reliability of the experimental methods that they presented and their mathematical model enabled further researchers to build off of their discovery, culminating with the patch-clamp method which has revolutionized the research of electrophysiology since the 1970s [5]. The ability for researchers to study nonexcitable cells ion channels as well as individual channels within neurons and other excitable cells has been a boon to researchers since the 1970s [5, 6].

The track from Galvani’s initial famous frog leg experiments to modern research using the patch-clamp is a testament to the resolve of science as an institution. Over 200 years have passed since Galvani’s initial experiments, simply involving the electrical stimulation of frog nerves, to being able to measure the individual ionic currents within those nerves. Research in an overarching field such as electrophysiology is not a fast process. The lesson to be learned from its success is that solid foundational work is necessary for the future improvements and successes in the field. Without the work of other researchers prior to Hodkgins and Huxley such as Cole and Marmots, the revolutionary isopotential membrane created by a dual-electrode voltage-clamp would not have happened. The revolutionary patch-clamp was built upon the earlier work of Hodgkins and Huxley, and this method has allowed electrophysiology researchers to expand into many different cell types.

 

 

References

 

[1] Schwiening CJ. A brief historical perspective: Hodgkin and Huxley. The Journal of Physiology. 2012;590:2571-5.

[2] Hodgkin AL, Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. The Journal of physiology. 1952;117:500.

[3] Hamill OP, Marty A, Neher E, Sakmann B, Sigworth F. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv European journal of physiology. 1981;391:85-100.

[4] Sakmann B, Neher E. Patch-clamp techniques for studying ionic channels in excitable membranes. Annual review of physiology. 1984;46:455-72.

[5] Veitinger DS. The Patch-Clamp Technique: An Introduction Science Lab by Leica Microsystems2011.

[6] Cuevas J. Electrophysiological Recording Techniques.  xPharm: The Comprehensive Pharmacology Reference. New York: Elsevier; 2007. p. 1-7.

 

Leave a Reply