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Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system
Biophysical Journal, Dec 2002 by Gorelik, Julia, Gu, Yuchun, Spohr, Hilmar A, Shevchuk, Andrew I, Et al
ABSTRACT We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipete is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.
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INTRODUCTION
The patch-clamp technique, first described by Neher and Sakmann (Neher and Sakmann, 1976; Neher et al., 1978) and initially refined by Hamill (Hamill et al., 1981) is the main experimental approach used for obtaining information about the characteristics and distribution of ion channels in living cells. Some variations of this technique have since been developed for specific experimental situations (Levitan and Kramer, 1990; Jonas et al., 1997), and together these methods have allowed recording in an enormous variety of situations. Data from this type of experiment, along with a host of other work, have shown that ion channels frequently associate with specific subcellular structures and are not uniformly distributed on the cell surface; this is important for their function (Joe and Angelides, 1992; Angelides, 1986; Banke et al., 1997; Alkondon, 1996; Tousson et al., 1989; Frosch and Dichter, 1992; Kinnamon et al., 1988; Karpen et al., 1992; Cohen et al., 1991; Gu et al., 2002; Korchev et al., 2000a) However, the available methods remain limited when it comes to studying this subcellular distribution of channels. For example, it is still difficult to record from fine structures such as microvilli and the fine dendritic branches of neurons or to patch opaque samples or obscured structures such as transverse tubules from muscle. A major reason for this is the difficulty in controlling patch pipettes in their approach to such samples. This is because an electrode is typically positioned using manual adjustments while focusing between the pipette and sample under a light microscope. Some samples, such as transverse tubules, are not optically visible so these present inherent difficulty. Ultra-fine structures also provide a considerable challenge because high-magnification objectives with a short depth of field are used so that for much of the pipette approach, sample and electrode are not in the same focal plane. Under these circumstances it is easy to damage the pipette tip.
In this study we demonstrate the utility of a new patch-clamp method that solves these problems and enables both the identification of small cellular or subcellular membrane structures as well as subsequent patch recording. Our method combines the capabilities of conventional patch clamping with the advanced features of scanning ion conductance microscopy (SICM) (Hansma et al., 1989). SICM allows both ultra-fine positioning of the probe over the sample and high-resolution imaging of living cell membranes (Korchev et al., 1997, 2000b; Shevchuk et al., 2001). Because the SICM probe is also used as the patch pipette, it provides its own image of the cell surface and ensures precise positioning of the electrode relative to the cell topography. The development of this method thus allows the investigation of ion channels that have a unique spatial distribution in otherwise difficult samples and may also lead to new methods for automated patch recording.
MATERIALS AND METHODS
Operation of the scanning patch-clamp is based on the idea that both SICM and patch-clamp recording use a glass micropipette as their probe. By combining these techniques the same micropipette can be used first in SICM protocols to image the cell surface and identify membrane structures of interest and then as a patch pipette for electrophysiological recording as shown schematically in Fig. 1. In this method the scanning micropipette is arranged vertically and manipulated by SICM computer control (Fig. 2 A). A feedback control system is in operation while the pipette approaches the cell surface. As soon as the pipette reaches a distance d from the surface, the SICM feedback control maintains a constant tip-sample separation. This procedure makes the approach straightforward and safe, because the patch pipette is prevented from touching the cell membrane until it is desired to do so to form a seal. It is important to note that a visual image of the sample is not necessary for this approach. Once the SICM protocol has obtained a topographic image of the cell surface it can be used to position the patch pipette over an exact place of interest for patch recording (Fig. 2 B). Finally, feedback control is switched off, the pipette is lowered, and suction is applied, resulting in the formation of a gigaohm seal (Fig. 2 C). Ion channel recording is then performed by conventional methods with all configurations available, e.g., cell-attached, inside-out, whole-cell, and/or outside-out mode.
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