Recovery from desensitization of neuronal nicotinic acetylcholine receptors of rat chromaffin cells is modulated by intracellular calcium through distinct second messengers

Abstract

The mechanisms through which changes in intracellular Ca2+ concentration ([Ca2+]i) might influence desensitization of neuronal nicotinic receptors (nAChRs) of rat chromaffin cells were investigated by simultaneous patch-clamp recording of membrane currents and confocal microscopy imaging of [Ca2+]i induced by nicotine. Increases in [Ca2+]i that were induced by membrane depolarization or occurred spontaneously did not influence inward currents elicited by focally applied test pulses (10 msec) of nicotine, indicating that raised [Ca2+]i per se did not trigger desensitization of nAChRs. Desensitization of nAChRs, evoked by 2 sec focal application of nicotine, which largely raised [Ca2+]i, was not affected by intracellular application of agents that activate or depress protein kinase C (PKC) or A (PKA) or inhibit phosphatase 1, 2 A and B. Conversely, recovery from desensitization was facilitated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the phosphatase 2 B inhibiting complex of cyclosporin A-cyclophilin A, whereas it was impaired by the broad spectrum kinase inhibitor staurosporine. The effects of PMA or staurosporine were prevented by the intracellularly applied Ca2+ chelator BAPTA. The adenylate cyclase activator forskolin accelerated recovery, whereas the selective PKA antagonist Rp-cAMPS had an opposite effect. The action of staurosporine and Rp-cAMPS on recovery from desensitization was additive. It is proposed that when nAChRs are desensitized, they become susceptible to modulation by [Ca2+]i via intracellular second messengers such as serine/threonine kinases and calcineurin. Thus, the phosphorylation state of neuronal nAChRs appears to regulate their rate of recovery from desensitization.

Fig. 1.

Combined recording (fluo-3-containing pipette) of membrane currents and [Ca²⁺]_i from chromaffin cells. A, The conditioning pulse of nicotine (2 sec; horizontal bar) produced a fading current response (b) and a persistent increase in [Ca²⁺]_i (a), with subsequent depression of test current amplitude induced by 10 msec nicotine pulses (b, arrowheads) attributable to desensitization of nAChRs. Break in trace corresponds to 30 sec. B, A 0.5 sec depolarization of membrane potential (from −70 to 0 mV) induces a relatively large elevation in [Ca²⁺]_i (thick trace in Ba) associated with a transient inward current (see Bc). When a similar depolarization is applied (see *) 2 sec before the test pulse of nicotine (thin trace, arrowheads), no depression of the nicotine-induced current (b) is present. Thirty seconds later, test current amplitude is also not different from control.C, Average time course of recovery from desensitization (evoked by 2 sec nicotine application) obtained from a sample of 10 cells in control conditions can be fitted with an exponential function (τ = 44 sec). D, Average amplitude of test currents 2 and 30 sec after voltage step is not different from control (n = 11).

Fig. 2.

Spontaneous [Ca²⁺]_i rise does not affect amplitude of test nicotine currents. A, Combined recording of [Ca²⁺]_i (a) and membrane currents (b) induced by 10 msec test applications of nicotine (short arrows) before (left), during (middle), or after (right) the spontaneous increase in [Ca²⁺]_i (indicated by the long arrow in a). Note onset of the persistent inward current (arrow in b) accompanying the [Ca²⁺]_i increase. B, Data pooled from 7 cells indicate lack of change in the normalized amplitude of test currents close to the peak of, or 30 sec after, the spontaneous [Ca²⁺]_i rise.

Fig. 3.

Effects of the phorbol ester PMA or staurosporine on recovery from desensitization of nAChRs. A, Membrane currents (b) and [Ca²⁺]_i transients (a) evoked by 10 msec test pulses (arrows) of nicotine applied every 15 sec from cell patched with a PMA (100 nm)-containing pipette. Note that despite a persistent increase in [Ca²⁺]_i, large recovery of current amplitude already takes place 15 sec after the desensitizing dose of nicotine. B, Similar protocol applied to cell patched with a pipette containing 100 nm staurosporine reveals incomplete recovery from desensitization despite return of [Ca²⁺]_i elevated by the 2 sec nicotine pulse to baseline. C, Data pooled from 8–14 cells recorded with control (squares), PMA-containing (upward triangles), or staurosporine-containing (staur; downward triangles) pipette solution. *, ** =p < 0.03 or 0.001, respectively. D, In parallel experiments intracellular application of 10 mmBAPTA prevents effects of PMA or staurosporine, revealing their [Ca²⁺]_i dependence (n = 7–11).

Fig. 4.

Acceleration of recovery from desensitization by the selective calcineurin antagonist CC complex. A, Membrane currents (b) and [Ca²⁺]_i transients (a) simultaneously recorded from a cell patched with CC complex (20 nm)-containing pipette. Note fast recovery of test current amplitude after the conditioning pulse of nicotine. B, Data pooled from three cells indicate an increase in recovery rate in the presence of CC complex (upward triangle). *, ** = p < 0.04 and 0.05, respectively.

Fig. 5.

Action of the PKA inhibitor Rp-cAMPS or the adenylate cyclase activator forskolin on recovery from desensitization. Membrane currents (b) and [Ca²⁺]_i transients (a) are shown in A andB. A, Standard protocol to induce desensitization applied to a cell patched with Rp-cAMPS (10 μm)-containing pipette reveals slow recovery.B, Opposite action of forskolin (10 μm) consisting of acceleration of recovery under the same experimental protocol. C, Pooled data summarizing recovery time course from 5–10 cells in control (squares) or in the presence of forskolin (upward triangles), Rp-cAMPS (downward triangles), or mixture of Rp-cAMPS and staurosporine [Rp + staur (100 nm); ×]. Note substantially slower recovery in the presence of both Rp-cAMPS and staurosporine. + = p < 0.0003; *, **, *** =p < 0.05, 0.001, or 0.003, respectively.