Double patch clamp reveals that transient fusion (kiss-and-run) is a major mechanism of secretion in calf adrenal chromaffin cells: high calcium shifts the mechanism from kiss-and-run to complete fusion

Abstract

Transient fusion ("kiss-and-run") is accepted as a mode of transmitter release both in central neurons and neuroendocrine cells, but the prevalence of this mechanism compared with full fusion is still in doubt. Using a novel double patch-clamp method (whole cell/cell attached), permitting the recording of unitary capacitance events while stimulating under a variety of conditions including action potentials, we show that transient fusion is the predominant (>90%) mode of secretion in calf adrenal chromaffin cells. Raising intracellular Ca2+ concentration ([Ca]i) from 10 to 200 microM increases the incidence of full fusion events at the expense of transient fusion. Blocking rapid endocytosis that normally terminates transient fusion events also promotes full fusion events. Thus, [Ca]i controls the transition between transient and full fusion, each of which is coupled to different modes of endocytosis.

Figure 1.

DMPP triggers burst and isolated single secretory events. A, Real and imaginary traces, evoked by 10 μm DMPP, correspond, respectively, to the conductance and capacitance of the patch under a cell-attached pipette, as shown in the inset. N, Nucleus. B, Fusion pore conductance (G_p) and vesicle capacitance (C_v) of transient fusion events are calculated from real and imaginary traces as follows: G_p = (Re² + Im²)/Re; C_v = [(Re² + Im²)/Im]/ω (see Materials and Methods). C, Histogram of the C_v, G_p, and event duration (n = 169; 18 cells) with their corresponding mean values. When DMPP was omitted from the pipette solution, from 141 cells tested, only 6 cells had a total of three transient fusion events and seven full fusion events (data not shown), indicating that cells were primarily quiescent before stimulation. DMPP triggered the first event with a mean delay time of 167 ± 28 s (see Materials and Methods). Increasing DMPP to 100 μm did not decrease the delay time or increase the number of events. Re, Real; Im, imaginary.

Figure 3.

High [Ca]_i switches the mode of fusion from transient to full. A, A concentration of 10 μm free [Ca]_i, dialyzed into the cell through the whole-cell patch pipette, induces a continuous increase in the whole-cell membrane capacitance (C_m) (a₁) at a rate of 8.1 ± 0.6 fF/s (n = 13 cells) and transient fusion events in the cell-attached recording (a₃). A stimulation protocol (10 × 50 ms) was applied to the cell 1 min after establishing the whole-cell configuration and every 5 min thereafter to track the effect of [Ca]_i on RE kinetics. B, A concentration of 102 μm free [Ca]_i triggered a continuous increase in the whole-cell C_m (b₁) at a rate of 13 ± 0.7 fF/s (n = 11 cells) and evoked both transient fusion and full fusion events in cell-attached traces (b₃). RE is still intact 1 min after getting into the whole-cell recording but compromised at 6 min (b₂). C, A concentration of 210 μm free [Ca]_i triggered a continuous increase in the whole-cell C_m (c₁) at a higher rate of 23.5 ± 2 fF/s (n = 11 cells) and evoked mostly full fusion events in the cell-attached recording (c₃). RE is abolished under these conditions at 1 and 6 min (c₂). Note that RE is compromised by the second stimulation at ∼102 μm [Ca]_i (a₂) and blocked by the following third or fourth stimulations (data not shown).

Figure 2.

APs trigger predominantly transient fusion events. A, Diagram showing the double patch-clamp approach: the left patch pipette in the cell-attached configuration (CA) to record capacitance and conductance of single secretory events and the right pipette in the whole-cell configuration (WC) to stimulate the cell with APs. N, Nucleus. B, Conductance (Re) and capacitance (Im) traces show transient fusion events evoked by APs shown underneath at 1 and 7 Hz. C, Histograms of the mean values of the C_v, G_p, and event duration of the transient events evoked by APs at 1 Hz (69 events, 15 cells) and 7 Hz (150 events, 19 cells). Each cell was stimulated with different rounds of 150 or 500 APs, separated by a 4 min resting period, at 1 or 7 Hz, respectively.

Figure 4.

High [Ca]_i shifts the mode of secretion to full fusion. A, Different levels of free Ca²⁺ concentrations were fixed with different Ca²⁺ buffers (see Materials and Methods) and introduced into the cell through the whole-cell pipette. The occurrence probability of secretory events showing a shift toward full fusion at the expense of transient fusion events at a higher level of [Ca]_i is shown. The numbers in parentheses are the number of full fusion events in each condition. B, Analysis of a full fusion event shown at an expanded time. C_v = Im/ω and the conductance of the fusion pore is calculated from the imaginary trace (Im) alone, G_p = Im/(Im/(Im − 1))^^1/2 (see Materials and Methods). The G_p was calculated only when the fusion pore stabilized for a few milliseconds (410 pS) before it reached high values (∼1 nS). The expansion of the fusion pore is expected to be accompanied by a transient increase in the real trace (Re).

Figure 5.

Switch from kiss-and-run to full fusion by blocking RE. A, A stimulation protocol (10 × 50 ms) triggers an increase in the membrane capacitance (C_m), exocytosis, coupled to RE (a₁). In the mean time, one kiss-and-run event of 1.8 fF was seen in the cell-attached recording after the third stimulation (a₂). The whole-cell C_m is the balance between exocytosis and endocytosis that is in favor of exocytosis during stimulations and in favor of endocytosis at the end of stimulation. B, When bath Ca²⁺ was substituted for Sr²⁺ and the cell-attached pipette Ca²⁺ was substituted for Sr²⁺, the whole-cell C_m evoked by 10 × 50 ms pulses shows the C_m increase without the ensuing RE at the end of the pulses (b₁). In the mean time, three full fusion events were recorded in the cell-attached trace (Im/ω; b₂) after the end of stimulation. The conductance trace (Re) shows a brief transient before the fusion pore expands fully. C, Whole-cell membrane capacitance (C_m), evoked by 29 × 75 ms pulses in the presence of Ca²⁺, manifests as a robust C_m increase, followed by SE (c₁). Concurrently, two full fusion events were evoked in the cell-attached capacitance recording (Im/ω; c₂), at the 20th and 21st pulses generated in the whole-cell pipette. The dashed lines represent a lack of capacitance recording during the pulses.