Kinetics of desensitization and recovery from desensitization for human α4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells
Abstract
Aim: Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human α4β2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line.
Methods: Whole-cell patch clamp recordings were performed to evaluateα4β2-nAChR currents.
Results: Application of 0.1 μmol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of ~70 and ~700 ms, for the onset of α4β2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of α4β2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs1 from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human α4β2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings2 using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse.
Conclusion: These findings indicate the existence of α4β2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.
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Methods: Whole-cell patch clamp recordings were performed to evaluateα4β2-nAChR currents.
Results: Application of 0.1 μmol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of ~70 and ~700 ms, for the onset of α4β2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of α4β2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs1 from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human α4β2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings2 using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse.
Conclusion: These findings indicate the existence of α4β2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.