Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromaffin cells

Abstract

1. Endocytosis following exocytosis evoked by single step depolarizations was examined in bovine adrenal chromaffin cells using high resolution capacitance measurements in perforated-patch voltage clamp recordings. 2. Endocytosis was detected as a smooth exponential decline in membrane capacitance to either the pre-stimulus level ('compensatory retrieval') or far below the pre-stimulus level ('excess retrieval'). During excess retrieval, > 10% of the cell surface could be internalized in under 5 s. 3. Compensatory retrieval was equal in magnitude to stimulus-evoked exocytosis for membrane additions > 100 fF (about fifty large dense-cored vesicles). In contrast, excess retrieval surpassed both the stimulus-evoked exocytosis, and the initial capacitance level recorded at the onset of phase-tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly returned to pre-stimulus levels, even in the absence of stimulation. 4. A large percentage of capacitance increases < 100 fF, usually evoked by 40 ms depolarizations, were not accompanied by membrane retrieval. 5. Compensatory retrieval could occur with any amount of Ca2+ entry, but excess retrieval was never triggered below a threshold Ca2+ current integral of 70 pC. 6. The kinetics of compensatory and excess retrieval differed by an order of magnitude. Compensatory retrieval was usually fitted with a single exponential function that had a median time constant of 5.7 s. Excess retrieval usually occurred with double exponential kinetics that had an extremely fast first time constant (median, 670 ms) and a second time constant indistinguishable from that of compensatory retrieval. 7. The speed of compensatory retrieval was Ca2+ dependent: the largest mono-exponential time constants occurred for the smallest amounts of Ca2+ entry and decreased with increasing Ca2+ entry. The Ca2+ dependence of mono-exponential time constants was disrupted by cyclosporin A (CsA), an inhibitor of the Ca(2+)- and calmodulin-dependent phosphatase calcineurin. 8. CsA also reduced the proportion of responses with excess retrieval, but this action was caused by a shift in Ca2+ entry values below the threshold for activation. The lower total Ca2+ entry in the presence of CsA was due to an increase in the rate of Ca2+ current inactivation rather than a reduction in peak amplitude. 9. Our data suggest that compensatory and excess retrieval represent two independent, Ca(2+)-regulated mechanisms of rapid membrane internalization in bovine adrenal chromaffin cells. Alternatively, there is a single membrane internalization mechanism that can switch between two distinct modes of behaviour.

Figure 1. Decays in capacitance following single depolarizations can be fitted with either a single exponential function or the sum of two exponentials

C_m traces from 2 cells stimulated by 320 ms depolarizations from -90 to +20 mV (gap in capacitance recording occurs during depolarization). Exponential fits of capacitance decays were carried out in Microcal Origin (see Methods). In A and B, single (continuous curves) and double (dashed curves) exponential fits of the same capacitance decay are shown superimposed for the two endocytotic responses. A, a single exponential fit deviated substantially from the observed decay in capacitance, but the capacitance decrease was well fitted with a double exponential function. B, a double exponential fit of the capacitance decay was not visually distinguishable from a single exponential fit; the single τ and τ₁ from the double exponential function differed < 2-fold. Ca²⁺ current integrals were 94.4 pC and 121 pC for cells in A and B, respectively. (Cell L060401 and L092403.)

Figure 3. Excess retrieval and compensatory retrieval: two types of endocytosis that can occur in an individual bovine adrenal chromaffin cell

Plot of cell capacitance throughout a 25 min recording period. Stimulation protocols are indicated with symbols; above is the total Ca²⁺ entry (Q_Ca) for each protocol. The cell was stimulated with 3 single long duration depolarizations (320, 320 and 640 ms), and each evoked large C_m jumps that were followed by decreases in capacitance. Endocytosis, after the first two long depolarizations, rapidly undershot the pre-stimulus C_m level by >100 fF (Excess retrieval); the third long depolarization evoked exocytosis followed by compensatory retrieval back to the pre-stimulus level. Individual capacitance traces (duration ∼20 s) were aligned manually in Microcal Origin (see Methods); calibration pulses have been eliminated for clarity. (Cell L070908.)

Figure 6. Regulation of cell surface area during prolonged perforated-patch recordings

Continuous capacitance records, obtained by aligning single sequential traces as described in Methods. Capacitance acquisition was initiated when access conductance became > 70 nS. On a slow time scale, gradual stimulus-independent changes in cell capacitance become apparent. Cell 1 and Cell 3 both show slow upward drift in C_m at the beginning of the record that sums with C_m jumps evoked by 40 ms pulses, whilst Cell 2 has a slow C_m decline. The first 320 ms pulse depolarization often triggered rapid and large amplitude excess retrieval that could truncate (Cells 1 and 2) or even obliterate (Cell 3) the C_m jump. After excess retrieval, C_m did not normally remain at the new level but instead increased slowly, appearing to approach the initial or pre-stimulus level. This post-excess retrieval increase in capacitance could occur in the absence of depolarization- induced Ca²⁺ entry (Cell 1). Only 1 cell out of 8 that exhibited excess retrieval (11 continuous plots were assembled) remained more or less at the level reached by the excess retrieval event (Cell 2). Excess retrieval tended to become smaller with repeated stimulations (compare responses to 320 or 640 ms depolarizations, early vs. late in the recordings). Stimulation protocols: □, single 40 ms pulse; ▵, single 160 ms pulse; ○, single 320 ms pulse; ⋄, single 640 ms pulse; ⋆, train of 5 ms pulses (35); ×, train of 40 ms pulses (20). (Cells L060204, L091101, L081902.)

Figure 2. Capacitance decays following single depolarizations are not significantly obscured by overlapping exocytosis

A, capacitance recording (C_m) and simultaneous amperometric recording (I_amp) from a cell depolarized with a single 640 ms depolarization. The arrow indicates where the initial limit would be set for fitting the decay with an exponential function. The pre-stimulus level (dotted line) was reached ∼15 s after the depolarization (not shown). B, an expansion of the amperometric current trace in A, during and 300 ms following the depolarizing pulse. There are 8 distinct current spikes during the depolarization; one large spike occurs at ∼150 ms after the depolarization. The Ca²⁺ current evoked by the depolarization is shown below on the same time scale; note the almost complete inactivation of current by the end of the pulse. The Ca²⁺ current integral is 120 pC. (Cell L061701.) C, summary histogram of the timing of amperometric current spikes evoked by 320 ms pulses (n= 6 stimulations, 4 cells) and 640 ms pulses (n= 2 stimulations, 2 cells). The x-axis indicates time elapsed from the onset of the depolarization; bin width, 25 ms. The end of the depolarization is indicated by an arrowhead.

Figure 4. Compensatory retrieval, following brief and long duration pulses, within individual bovine adrenal chromaffin cells

Cell 1, left; a 40 ms depolarization from -90 to +20 mV evoked a 50 fF C_m jump, followed by slow endocytosis that decayed to within 10 fF of the pre-stimulus level (dotted line) in ∼60 s (exponential fit is superimposed as a continuous curve). Inset, inward current trace; integrated Ca²⁺ entry, 17.3 pC (all inward current traces in this figure are shown on the same time scale). Cell 1, right; a 320 ms depolarization evoked an ∼150 fF increase in C_m that rapidly decayed with double exponential kinetics (continuous curve) to slightly past the pre-stimulus level within a single C_m trace (total duration, ∼20 s). Inset, inward current trace; integrated Ca²⁺ entry, 114 pC. (Cell L071802.) Cell 2, left; after a small C_m jump evoked by a 40 ms depolarization, C_m remained at approximately the same level for at least 20 s; this trace could not be fitted by the exponential-fitting algorithm and was judged as having no endocytosis. Cell 2, right; an ∼120 fF C_m jump elicited by a 160 ms depolarization was followed by a mono-exponential decline to the pre-stimulus level within a single C_m trace (fit shown superimposed as a continuous curve). Total Ca²⁺ entry was 25.9 pC and 82.9 pC for the 40 ms and 160 ms pulses, respectively (see insets). (Cell N040101.)

Figure 5. Compensatory retrieval is accurate and reliable for large C_m jumps

A, the magnitude of endocytosis vs. the C_m jump for single depolarizations (40-640 ms duration; symbol for each duration as indicated). Total endocytosis in femtofarads was determined from the amplitude of the best exponential fit to the C_m decay (A, or A₁+A₂; see Methods) whilst the C_m jump was calculated from the average across the first 10 capacitance points subsequent to the depolarization. Therefore some compensatory endocytotic responses are significantly larger than the C_m jump, because membrane added after the time used for C_m jump calculation was retrieved accurately (i.e. ⋄, 550 fF at exocytosis, 780 fF at endocytosis). Excess retrieval events (undershoot > 100 fF) were excluded from analysis. Inset, expansion of the region 0-100 fF. Many C_m jumps in this region were not accompanied by endocytosis (decay undetectable based on inability to be fitted by an exponential function). B, stimulus-evoked responses were sorted independently by (a) exocytosis (i.e. size of C_m jump) or (b) Ca²⁺ entry, and the percentage of responses accompanied by endocytosis was calculated for each bin.

Figure 7. Excess retrieval events following single step depolarizations

A, excess retrieval after a single 320 ms depolarization to +20 mV (from a holding potential of -90 mV; see inset for inward current trace) that undershoots the pre-stimulus level (dotted line) by 213 fF. The decay was well fitted by a single exponential (dashed curve). Total Ca²⁺ entry was 120 pC. (Cell N020302.) B, excess retrieval could be extremely rapid, reaching a maximum undershoot hundreds of femtofarads below the pre-stimulus level in under 5 s (undershoot, 440 fF). This large undershoot represented a decrease in total cell capacitance as it required a readjustment of the capacitance compensation circuitry. The endocytotic response was fitted with the sum of two exponentials (dashed curve), as were most rapid excess retrieval events. Inset: total Ca²⁺ entry, 320 ms depolarization, 143 pC. (Cell N120502.) The accompanying conductance traces (G) do not show parallel equal (or opposite) changes.

Figure 8. Excess retrieval requires a threshold amount of Ca²⁺ entry for activation

A, plot of the undershoot (in femtofarads, relative to pre-stimulus baseline) as a function of total Ca²⁺ entry, for the first long duration (≥ 160 ms) stimulation of an experiment. A point for the 40 ms stimulation immediately prior to the long duration stimulation is also included for each cell. The shaded region covers undershoots ≤ 100 fF that are classified as compensatory retrieval. No excess retrieval occurred if the Ca²⁺ current integral was ≤ 70 pC (arrow). B, endocytotic responses to 320 ms pulses were sorted by amount of Ca²⁺ entry and classified as either compensatory or excess retrieval. The percentage of responses with excess retrieval increased with increasing amount of Ca²⁺ entry, to a maximum of ∼70 %.

Figure 9. The distribution of time constants for endocytotic events is Ca²⁺ dependent

A, distributions of first (a) and second (b) time constants from double exponential decays, and single time constants from mono-exponential decays (c). Data include excess and compensatory responses, and all stimulations throughout the experiment. Note axis break between 15 and 30 s, so that the wide range of values could be displayed on the same plot. B, for a fixed pulse duration (320 ms) the majority of excess retrieval events were fitted with a double exponential function (59/69; 85 %). Only a fraction of responses with compensatory retrieval required two exponentials for a good fit (11/40; 27.5 %). C, Ca²⁺ dependence of single exponential time constants. Single τ values were binned by amount of Ca²⁺ entry and plotted as means ±s.e.m. (•) and medians (⋆) for each bin. The x-axis error bars represent the means ±s.e.m. for the range of Ca²⁺ entry covered by a particular bin.

Figure 10. CsA decreases total Ca²⁺ entry and inhibits exocytosis

A, inward current traces before and after perfusion with 1 μM CsA. a, CsA had little effect on peak current, but increased the rate of inactivation during the pulse. The change in inactivation rate developed slowly during continued perfusion with CsA, as illustrated by currents evoked by 40 ms pulses given prior to (-4 min), and 8 and 15 min after the perfusion started. b, in the same cell, the effect on inactivation was more apparent for currents evoked by 320 ms pulses, which were given once before (-7 min), and once after (17 min CsA) CsA had been applied for at least 15 min. In control, untreated cells there was no change in rate of inactivation for currents evoked by 320 ms depolarizations given > 15 min apart (not shown), although in some cells the peak current amplitude declined slightly during the course of an experiment. c, comparison of total integrated Ca²⁺ entry for 320 ms depolarizations in the absence of CsA (n= 28) vs. integrated Ca²⁺ entry for 13 responses after treatment with CsA for at least 15 min (*; P < 0.05, Student's t test). B, plot of C_m jumps, binned by amount of Ca²⁺ entry, for control responses and responses elicited in the presence of CsA. A standard curve obtained from the average of 27 Ca²⁺-secretion relationships obtained in a previous study (Engisch & Nowycky, 1996) is superimposed on the data (dashed curve). Control responses (□) lie close to the standard curve. However, C_m jumps after CsA treatment (▴) are significantly less than control responses at the highest Ca²⁺ entry range attained in the presence of CsA (P < 0.05, Student's t test).

Figure 11. Compensatory retrieval is slower and less accurate after CsA application

A, four examples of relatively large C_m jumps (close to or > 100 fF) elicited in the presence of CsA. Three sequential C_m traces were aligned for each response shown. After CsA treatment, C_m decays were slow and often incomplete. a and b, (cell L073002) response to 160 ms depolarization (integrated Ca²⁺ entry, 77 pC) and 320 ms depolarization (110 pC), respectively; c, (cell L091102) response elicited by 320 ms depolarization (75 pC); d, (cell L080102) response to a 320 ms depolarization (72 pC). B, after treatment with CsA, the time constants for mono-exponential decays no longer displayed any Ca²⁺ dependence. Time constants were binned by amount of Ca²⁺ entry; only the three lowest ranges were observed in the presence of CsA. Values for time constants in these ranges for control responses are replotted from Fig. 9 for comparison. At the highest range, the time constant was significantly different from control (P < 0.05, Mann-Whitney U test). C, effect of CsA treatment on excess retrieval. The undershoot (relative to pre-stimulus baseline) was plotted as a function of integrated Ca²⁺ entry for responses in the presence of CsA (▴) and control responses in the same experiments or from cells in sister cultures (○ data are a subset of the data presented in Fig. 8A). Shaded region covers undershoots ≤ 100 fF that are classified as compensatory retrieval. CsA treatment did not alter the threshold Ca²⁺ requirement of excess retrieval, but due to the increase in rate of inactivation, a higher proportion of 320 ms pulses induced Ca²⁺ entry that fell at or below the threshold amount (arrow).