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High-resolution scanning patch-clamp: new insights into cell function¹

YUCHUN GU², JULIA GORELIK², HILMAR A. SPOHR, ANDREW SHEVCHUK, MAX J. LAB^*, SIAN E. HARDING^*, IGOR VODYANOY^*,, DAVID KLENERMAN and YURI E. KORCHEV³

Division of Medicine, MRC Clinical Sciences Center, Faculty of Medicine, Imperial College of Science, Technology and Medicine;
^* National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London, UK;
Office of Naval Research, Arlington, Virginia, USA; and
Department of Chemistry, Cambridge University, Cambridge, UK

³Correspondence: Division of Medicine, MRC Clinical Sciences Center, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. E-mail: y.korchev{at}ic.ac.uk

SPECIFIC AIMS

Studies suggest that cell specialization is often governed by the unique spatial distribution of ion channels on the cell surface, but the functional localization of ion channels is not well known due to the lack of methods that allow imaging of topographical details of a living cell surface and simultaneous electrophysiological study of ion channels at precisely chosen locations. We wanted to develop a patch-clamp system with high spatial resolution so that before performing electrophysiological measurements, the same patch-clamp nanopipette could be used for imaging the cell surface and positioned precisely at the region of interest.

We aimed to determine the functional localization of calcium and chloride channels in the cardiac myocyte sarcolemma and to define the link between cardiomyocyte structure, ion channel distribution, and cell function.

PRINCIPAL FINDINGS

1. High-resolution scanning patch-clamp technique enables the study of ion channels in small cells and submicrometer cellular structures
A new technique was developed with the aim of performing patch-clamp at a specified position on the cell surface in contrast to conventional patch-clamping with light microscope-aided guidance, where the position of the pipette in respect to the cell topography cannot be finely controlled. This was accomplished by first using a nanopipette as a scanning probe to image the surface topography of a cell, then precisely placing the pipette on a defined area on the surface in order to investigate single-channel currents at that position. We call this method high-resolution scanning patch-clamp or ‘smart’ patch-clamp (Fig. 1 ).

View larger version (60K): [in this window] [in a new window]   Figure 1. L-type Ca2+ channel distribution in the cardiac myocyte sarcolemma: mapping of ion channels by the high-resolution scanning patch-clamp technique. A) To perform patch-clamp recording from different regions on the cardiac myocyte sarcolemma, an area is selected (white dotted square). The patch-clamp nanopipette with a backfill solution for investigating Ca2+ channels is used to image the cell surface topography controlled by SICM. B) Experimental topographic image of a representative rat cardiomyocyte membrane. Z-grooves, T-tubule opening, and characteristic sarcomere units are marked. C) Functional schematic of sarcomere units showing the position of the probed region (Z-groove, T-tubule opening, and scallop crest). Probabilities of forming a G seal as a function of surface position shown in parentheses. D) Cell-attached Ba2+ current transients at voltages of + 20, ± 0, -20 mV. E) Several current transients elicited at 0 mV from one patch and ensemble average of 12 transients showing typical L-type inactivation kinetics. F) Statistical distribution of L-type Ca2+ channels with the highest density near the T-tubule opening.

Topographic imaging of the cell was performed using previously described scanning ion conductance microscopy methods. The pipette mounted on a piezo stage is moved over the cell maintaining a fixed distance to the surface. This is achieved by a feedback control keeping the ion current through the pipette constant. This setup was adapted for high-resolution patch-clamping by replacing the current amplifier with a commercial patch-clamp amplifier. Nanopipettes were made from borosilicate glass capillaries using a laser-based puller. Pipettes were used without any further treatment such as fire polishing or sylguard shielding. The pipette tip radius was 100 nm.

The pipette was vertically lowered by piezo control until contact with the cell surface. In T-tubule regions of living cardiomyocytes, a downward movement of up to 2 µm was required. Finally, light suction was applied to form a G seal. Since the patch is performed by a vertical approach controlling the pipette’s distance electromechanically, we found this method more reliable than conventional patch-clamping and less prone to variations between experimenters. The high success rate for obtaining patches allowed efficient accumulation of data points and statistical averaging of the channel distribution at distinct locations on the cell surface.

We investigated cellular structures of very small dimensions that are invisible under the light microscope, where patch-clamping has not been possible before. We investigated the Cl^- and Ca²⁺ channels in specific regions of the cardiomyocyte sarcolemma (Z-grooves, T-tubule opening, and scallop crest). We used the smart patch-clamp method on epithelial kidney cells and obtained cell-attached recordings of Cl^- channels from the top of microvilli.

Other examples of cellular structures of small size are neuronal dendrites and sperm cells. Their dimensions have previously rendered direct electrophysiological recordings unfeasible. We have applied the scanning patch-clamp technique to hippocampal neurons and superior cervical ganglion cells. It was found that K⁺ and Ca²⁺ channel currents can be measured on very fine dendrites ranging from 100 to 200 nm. Using scanning patch-clamp, we achieved high success rates for cell-attached recording on sea urchin sperm cells and human sperm cells, demonstrating an abundance of L-type Ca²⁺ channels on the cell body.

2. L-type Ca²⁺ channels are located in the T-tubule region of the cardiac myocyte sarcolemma
We identified and mapped the distribution of L-type Ca²⁺ channels in rat cardiac myocytes. Figure 1B shows a representative topographic image of the cell surface. The sarcomeres, openings of transverse tubules (T-tubules), and Z-grooves can be easily identified. A total of 233 patches were performed probing three different regions of the cardiomyocyte sarcolemma: the T-tubule openings, Z-grooves, and scallop crests (Fig. 1C ). Ion currents in cell-attached configuration were detected only when the pipette was located in the T-tubule opening. Current traces shown in Fig. 1D and current-voltage curves (not shown) are characteristic of L-type Ca²⁺ channels. Several single-channel Ca²⁺ currents of one patch are shown in Fig. 1E as well as the ensemble average, which identifies them as L-type. One of every eight patches in the T-tubule openings exhibited L-type Ca²⁺ currents, and these were not found in any other probed regions (Fig. 1F ). Other studies suggest that L-type Ca²⁺ channels are distributed mainly in the membrane of the T-tubule system. Using the probability of obtaining a patch containing calcium channels (P=0.125) and estimating the membrane patch area (0.06 µm²), based on the pipette geometry and supposing a hemispherical shape of the membrane patch, we estimated the density of L-type Ca²⁺ channels at T-tubule openings to be 2 channels/µm². Similar densities have been found in guinea pig cardiomyocytes using immunogold labeling.

3. Three types of Cl^- channels are located in the regions of T-tubule openings and Z-grooves
Three types of Cl^- currents were distinguished on the basis of single-channel conductance and rectification in cell-attached mode. Channels were identified as being Cl^- conductive from the reversal potential of the current-voltage curves. It was taken into account that any K⁺ currents were blocked by TEA and impermeable NMDG⁺ was the only cation present in the pipette. Two current types exhibited similar inward rectification and remained active after pulling the pipette off the cell to obtain the inside-out configuration. Channels of these subtypes may belong to the ClC family since their current-voltage characteristics resembled those of ClC channels in other tissue and types of cardiomyocytes. Expression of ClC-2 and ClC-3 channels in rat ventricle cells had already been demonstrated, but the lack of information on ion current characteristics in rat cardiac myocytes did not allow definitive identification before. A third type of current was found to be outwardly rectifying and resembled currents described in rabbit cardiomyocytes using conventional patch-clamp techniques. A total of 305 patches were performed at three distinct positions on the cell surface: the scallop crest, Z-groove, and T-tubule opening. All three types of Cl^- channels are distributed only in the regions of Z-grooves and around the T-tubule openings, but not on the scallop crest (Fig. 2 ).

View larger version (35K): [in this window] [in a new window]   Figure 2. Distribution of Cl- channels at the openings of the T-tubule system and colocalization with voltage-sensitive Ca2+ channels can provide precise, localized control over action potential propagation (APP) along the T-tubule system.

CONCLUSION AND SIGNIFICANCE

Smart patch-clamp
We have introduced a novel method for the high-resolution localization of single ion channels on a living cell surface. The ‘smart’ patch technique produces topographical images of 100 nm resolution and enables patch-clamp recording on very small membrane features invisible under the light microscope. It can be used on any functional ion channel without needing to know the molecular identity of the channel proteins examined. Conventional antibody staining techniques often require cells to be fixed and do not provide functional characteristics of the obtained ion channel distribution. The technique can be applied to small structures where patch-clamping has been difficult or impossible to perform and may be used for a wide range of cell types such as muscle cells, epithelial cells, neurons, and sperm cells. It is a robust and reliable method to perform patch-clamping. Potential applications of the technique include mapping of ligand-gated or mechano-sensitive ion channels, as the nanopipette can be used to deliver defined chemical, electrical, or mechanical stimuli to narrowly defined areas on the cell surface.

Ca²⁺ and Cl^- channels are colocalized at the T-tubule opening
Our results show that L-type Ca²⁺ channels are located only in the T-tubules and that Cl^- channels are clustered in the narrow regions of T-tubule openings and Z-grooves (Fig. 2) . There are no channels detected on the scallop crest. To determine whether our failure to observe currents on the scallop crest regions of the sarcolemma was an experimental artifact, a control experiment was carried out by inserting single -toxin channels into these regions. We successfully recorded -toxin-induced ion currents after such insertions. This confirmed that ion channel currents could in fact be observed at this position.

That the identified ion channels are confined to specific regions of the cardiomyocyte sarcolemma suggests they are most likely anchored by the cytoskeleton. Published results indicate a link between the F-actin cytoskeleton and L-type Ca²⁺ channels and Cl^- channels. Moreover, the channels are found in Z-grooves—sarcolemma regions that strongly interact with the intracellular cytoskeleton. It is from these regions that the T-tubules emanate into the interior of the cell forming the transverse tubular system of cardiac muscle. This system is a structure that allows rapid propagation of excitation into the cell interior, where during cardiac action potential Ca²⁺ enters the cell through depolarization-activated Ca²⁺ channels and triggers Ca²⁺ release from the sarcoplasmic reticulum. Indeed, we found that voltage-sensitive Ca²⁺ channels are distributed only in the T-tubular system and not in any other part of the cardiac myocyte sarcolemma. This strongly suggests that depolarization-activated Ca²⁺ entry into the cell can exclusively occur at the T-tubule system of ventricular myocytes.

Whereas Ca²⁺ is considered the most essential ion in the process of heart cell contraction and relaxation, the roles of Cl^- currents are presumed in shaping the cardiac action potential. Since Cl^- equilibrium potential is negative to plateau potentials and positive to the resting potential, Cl^- currents can modulate action potential. They can accelerate repolarization after the plateau phase, depolarize the cell, and prevent hyperpolarization of the cell. Cl^- currents thereby affect all important action potential phases and are relevant in automaticity and mechanisms of arrhythmia. In view of these facts and our own observations that Cl^- channels are localized only at the openings of the T-tubule system and colocalized in these regions with voltage-sensitive Ca²⁺ channels, we hypothesize that this proximity is crucial for electric coupling of these channels and necessary for providing a precise, localized, and as yet unrecognized control over action potential duration and propagation along the T-tubule system. Our previous finding of K_ATP channels aggregating in the same region of the sarcolemma strongly implies that action potential control is indeed highly spatially organized.

In conclusion, the spatial colocalization of Cl^-, Ca²⁺, and K⁺ ion channels in the sarcolemma region at the entrances of the T-tubule system may provide new insight into functional, synergistic coupling between the channels. Further investigation of spatial distribution of other cardiac ion channels could elucidate the roles of spatial ion channel coupling in excitatory mechanisms.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1024fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-1024fje

² Both authors contributed equally to this manuscript.

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This Article Full Text (PDF) All Versions of this Article: 16/7/748 01-1024fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GU, Y. Articles by KORCHEV, Y. E. Search for Related Content PubMed PubMed Citation Articles by GU, Y. Articles by KORCHEV, Y. E.