Figure 1. Patch-clamp technique. The schematic shows a patch pipette pressed up against a cell before suction is applied to the inside of the pipette to promote tight sealing. After sealing most of the current generated in the patch will flow up into the pipette to be measured by the patch-clamp circuitry. The figure shows a schematic of the current-to-voltage converter in the head stage in which the feedback resistor (R_f) determines the voltage to current conversion gain according to V_output=–R_fi_p+V_ref, where V_output represents the output voltage, i_p represents the patch current and V_ref represents the reference voltage set by the experimenter.

Figure 2. High-resolution video images of a tightly sealed membrane patch and the patch current during brief steps of suction and pressure. (a) and (b), images of the patch before, during and after applying steps of suction (a) and pressure (b). (c) and (d), the patch current recorded in the same patch imaged in (a) and (b), respectively, in response to suction and pressure steps. Modified from Zhang Y and Hamill, OP (2000) On the discrepancy between the whole-cell and membrane patch mechano sensitivity in Xenopus oocytes Journal of Physiology, 523(1): 101–115.

Figure 3. The membrane patch changes morphology during repetitive mechanical stimuli. (a–d) show video images of a cell-attached patch at different times after formation of the tight seal. (e–f) show the current response at the time of images (a) and (d), respectively. Between each image, brief suction pressure/suction protocols pulses were applied causing the membrane to progressively move up the pipette and a clear space developed between the membrane and the underlying cytoplasmic structures. Accompanying this membrane blebbing the mechanosensitive current was reduced in size and fast kinetics. Modified from Zhang Y et al. (2000) Mechanically-gated channel activity in cytoskeleton-deficient plasma membrane blebs and vesicles from Xenopus oocytes Journal of Physiology, 523(1): 117–130.

Figure 4. Schematic representation of the procedures used to form the different patch-clamp configurations. To obtain the cell-attached patch configuration the pipette tip is pressed against the cell and suction applied to the pipette interior (top figures). To form the inside-out patch the pipette is pulled away from the cell leaving a vesicle tightly sealed in the tip. The pipette tip is then briefly passed through the solution–air interface, which acts to disrupt the outer membrane of the vesicle (right hand figures). To form the outside-out patch, stronger suction is applied to the cell-attached patch until it ruptures gaining access to the inside of the cell. The pipette is then pulled away from the cell leaving the outer membrane face facing out and exposed to the external bath solution. Modified from Hamill et al. (1981).

Figure 5. Patch-clamp recording can be used to identify different types of coupling between receptors and channels. Each panel shows a cell-attached patch with a specific type of coupling between the channel and receptor. (a) Intrinsic coupling in which the receptor and channel are part of the same molecule. (b) 2nd messenger coupling in which activation of the receptor results in synthesis or release of a soluble 2nd messenger that can diffuse significant distance to activate the channel. (c) Membrane delimited coupling in which a structural component of the receptor dissociates from the receptor and diffuses within the membrane to activate channels in close proximity. (d) Conformational coupling in which a structural change in the receptor is directly transmitted to the channel molecule. In (a), (b) and (c) inclusion of agonist in the pipette activates the channel but bath-applied agonist only activates the channel in (b) because in this case the soluble 2nd messenger is able to diffuse into the patch and reach the channel. In (d) agonist included in either the pipette or the bathing solution may fail to activate the channel in the patch because the process of tight sealing physically disrupts the coupling between receptor and channel.