Nature © Macmillan Publishers Ltd 1997 27. Lee, R. H., Lieberman, B. S. & Lolley, R. N. Retinal accumulation of the phosphoducin/T and transducin complexes in developing normal mice and in mice and dogs with inherited retinal degeneration. Exp. Eye Res. 51, 325–333. 28. Dizhoor, A. M. et al. Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science 251, 915–918 (1991). 29. Raport, C. J. et al. Downregulation of cyclic GMP phosphodiesterase induced by expression of GTPase-deficient cone transducin in mouse rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 35, 2932–2947 (1994). 30. Baylor, D. A., Lamb, T. D. & Yau, K.-W. Responses of retinal rods to single photons. J. Physiol. (Lond.) 288, 613–634 (1979). Acknowledgements. This work was supported by grants from the National Eye Institute (D.A.B.), the National Institute on Aging (M.I.S.), the National Institute of General Medical Sciences (R.L.D.), the Ruth and Milton Steinbach Fund (D.A.B., J.C.), The McKnight Endowment Fund for Neuroscience (D.A.B.), Research to Prevent Blindness (J.C., C.L.M.) and Lions’ of Massachusetts (C.L.M.). Correspondence and requests for materials should be addressed to D.A.B. (e-mail: dbaylor@leland. stanford.edu) or J.C. (e-mail: jeannie@hsc.usc.edu). letters to nature NATURE | VOL 389 | 2 OCTOBER 1997 509 The exocytotic event in chromaffin cells revealed by patch amperometry A. Albillos*, G. Dernick*‡, H. Horstmann*, W. Almers*, G. Alvarez de Toledo† & M. Lindau*‡ * Department of Molecular Cell Research, MPI f. Medical Research, D-69028 Heidelberg, Germany † Department of Physiology & Biophysics, University of Seville, E-41009 Sevilla, Spain ......................................................................................................................... In mast cells and granulocytes, exocytosis starts with the forma- tion of a fusion pore 1–3 . It has been suggested that neurotrans- mitters may be released through such a narrow pore without full fusion 4,5 . However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca 2+ -dependent exocytosis and its role in trans- mitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements 3,6 combined with electrochemical detection of catecholamines 7,8 , achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determina- tion of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at 3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, gen- erating a ‘foot’ signal 8 which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane. Patch amperometry combines capacitance measurements and amperometric detection of catecholamine in the cell-attached con- figuration by introducing a carbon-fibre electrode (CFE) into a patch pipette (Fig. 1a). Cell-attached capacitance measurements allow resolution of single fusion events on the size scale of synaptic vesicles 3,6 . Vesicles fusing in the patch secrete their contents into the pipette. The CFE detecting released catecholamine must thus be placed inside the pipette. Figure 1b shows a cell at the beginning of the experiment with a patch pipette containing a CFE positioned 6–7 m from the pipette tip. After pipette attachment, exocytosis of chromaffin granules was indicated by amperometric signals associated with capacitance steps: part of the recording is shown in Fig. 1d. At the end of the experiment, the morphology of the cell had changed (Fig. 1c), indicating that massive exocytosis had occurred not only in the patch but all over the cell surface. This cell was pretreated with phorbol myristate acetate (PMA) to increase the pool of readily releasable vesicles 9 . ‡Present address: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853-2501, USA. After sealing the patch pipette to the plasma membrane, exocy- totic events occurred spontaneously in active patches. After steps had ceased, patch depolarization did not induce further capacitance steps. Similarly, in patches showing no spontaneous activity, depo- larization did not stimulate any capacitance step or amperometric transient. Thus, sealing of the pipette is sufficient to stimulate exocytosis of all releasable granules in the patch. No exocytotic events were observed when the bath and pipette solution contained no Ca 2+ and 1 mM EGTA (14 patches), indicating that exocytosis is Figure 1 a, Arrangement of a CFE inside a patch pipette. I A , Ampermetric current; I C , sine wave current used to measure capacitance changes. b, c, Chromaffin cell with attached patch pipette containing CFE at the beginning (b) and end (c) of the experiment. d, Recording from this cell shows amperometric transients (top), associated capacitance steps (middle), and conductance trace (bottom) using a 20-kHz, 50-mV sine wave. C is capacitance trace, G is conductance trace. Events 1 and 6 (labelled) are analysed in more detail in Figs 2, 3. e, Capacitance step size distribution obtained in experiments without PMA preincubation. f, Bars: frequency distribution of granule profile diameter from electron microscopy of resting chromaffin cells. Lines: profile size distributions expected from the conversion of capacitance step sizes, assuming specific capacitances of 9 fF m -2 (black) or 6 fF m -2 (grey). g, Relation between amperometric current amplitude and capacitance step size. h, Relation between catecholamine content and granule volume.