Abstract
We describe a simple pipette holder which allows, within a single experiment, multiple exchanges of the solution inside “gigaseal” glass pipettes commonly used for electrical studies of single cells or isolated membrane patches. The design minimizes electrostatic and mechanical perturbations associated with perfusion by integrating into the holder a reservoir which is connected to a perfusion pipette fabricated from flexible, resilient quartz tubing. The tip of the perfusion pipette can be pulled to any diameter and positioned precisely within the main patch-pipette by sliding the reservoir along a guide in the holder. An open reservoir for suction driven solution exchange, and a closed reservoir for pressure driven solution exchange were developed. For the open system, the speed of solution exchange was studied as a function of the tip diameter of the perfusion pipette (∼22 s for a 40 μm tip diameter). Both systems were characterized using atrial myocytes (a) by examining the effects of intracellular applications of cAMP or of the catalytic subunit of protein kinase A on calcium currents in the whole cell recording mode and (b) by studying the effects of local applications of acetylcholine (ACh) on single channel currents in the isolated membrane patch mode.
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References
Cull-Candy SG, Parker I (1983) Experimental approaches used to examine single glutamate-receptor ion channels in locust muscle fibers. In: Sakmann B, Neher E (eds) Single channel recording. Plenum Press, New York, pp 389–400
Gadsby DC, Kimura J, Noma A (1985) Voltage dependence of Na/K pump current in isolated heart cells. Nature 315:63–65
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100
Hume JR, Giles W (1983) Ionic currents in single isolated bullfrog atrial cells. J Gen Physiol 81:153–194
Kameyama M, Hofmann F, Trautwein W (1985) On the mechanism of β-adrenergic regulation of the Ca channel in the guineapig heart. Pflügers Arch 405:285–293
Mitra R, Morad M (1985) A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. Am J Physiol 149:H1056-H1060
Momose Y, Giles W, Szabo G (1984) Acetylcholine-induced K+ current in amphibian atrial cells. Biophys J 45:20–21
Neher E, Sakmann B (1976) Single channel currents recorded from membrane of denervated frog muscle fibers. Nature 260:799–802
Osterrieder W, Brum G, Hescheler J, Trautwein W, Flockerzi V, Hofmann F (1982) Injection of subunits of cyclic AMP-dependent protein kinase into cardiac myocytes modulates Ca2+ current. Nature 298:576–578
Sakmann B, Trube G (1984) Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart. J Physiol 347:641–657
Sakmann B, Noma A, Trautwein W (1983) Acetylcholine activation of single muscarinic K+ channels in isolated pacemaker cells of mammalian heart. Nature 303:250–253
Soejima M, Noma A (1984) Mode of regulation of the ACh-sensitive K-channel by the muscarinic receptor in rabbit atrial cells. Pflügers Arch 400:424–431
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Lapointe, JY., Szabo, G. A novel holder allowing internal perfusion of patch-clamp pipettes. Pflugers Arch. 410, 212–216 (1987). https://doi.org/10.1007/BF00581918
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DOI: https://doi.org/10.1007/BF00581918