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Department of Physiology and Biophysics, University of Washington, Seattle 98195.
1. Whole-cell and single-channel voltage-clamp techniques were used to record the 5-HT3 receptor-mediated currents in neurons freshly dissociated from rat superior cervical ganglia. 2. Whole-cell currents elicited by brief pressure ejection of 5-HT (10 microM) reversed at -4.5 mV when extracellular and intracellular solutions mainly contained NaCl and CsCl. The peak current-voltage relation showed modest inward rectification that was fully developed within less than 2 ms of the applied voltage step. 3. With prolonged application of 5-HT (10 microM) using a fast perfusion system, the response desensitized in two phases with fast and slow time constants of 0.57 and 6.0 s at -74 mV. The time constants showed little voltage dependence; however, the relative amplitude of the two components was significantly dependent on voltage. The time course of desensitization was not affected by agents that increase the levels of intracellular cyclic AMP. 4. The relative permeability of the channel was determined from reversal potential changes. The channel passed small cations non-selectively, with permeability ratios (PX/PNa) of 0.93 and 1.24 for Cs+ and K+. The organic cations Tris and glucosamine were measurably permeant with permeability ratios of 0.19 and 0.06. Ca2+ was fairly permeant with a relative permeability of 0.55 in 20 mM solution and of 0.16 when the concentration of CaCl2 was increased to 115 mM. No permeability was detected for Cl-. 5. Fluctuation analysis of the whole-cell current revealed an apparent single-channel current of approximately 0.18 pA at -74 mV. 6. 5-HT-activated single-channel currents were recorded in excised outside-out patches. When 5-HT (10 microM) was delivered by pressure ejection, channel openings appeared rapidly with a delay of 28 ms. The unitary current was about approximately 0.80 pA at -74 mV. The channel activity induced by bath perfusion of 5-HT (0.8 microM) was significantly reduced by 100 nM of the 5-HT3 receptor-specific antagonists 3-tropanyl-3,5-dichlorobenzoate (MDL 72222) or 3-tropanyl-indole-3-carboxylate (ICS 205-930). 7. The single-channel current-voltage relation was non-linear, with moderate inward rectification similar to that of the whole-cell current. The chord conductance of the channel decreased with membrane depolarization from 14.6 pS at -104 mV to only 9.9 pS at -54 mV. Open-time distributions consisted of two components with mean time constants of 0.45 and 2.8 ms at -104 mV. Burst-length distributions were also made up of two components with time constants of 0.45 and 4.6 ms.(ABSTRACT TRUNCATED AT 400 WORDS)
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