Kv1.1 potassium (K2؉ . In contrast, these treatments had little or no effect on the Lec mutants, which indicates that channel sialic acids appear to be the negative surface charges sensitive to Ca 2؉

No abstract

Previous studies have shown that the number of functional acetylcholine receptors (AcChoRs) on chicken ciliary ganglion neurons in culture is considerably smaller than the total number of AcChoRs detected on the neurons by labeled receptor probes. Here we use patch-clamp recording to show that a cAMP-dependent process enhances the AcCho response of the neurons by a mechanism likely to involve an increase in the number of functional AcChoRs. The increase occurs without requiring protein synthesis and without involving a detectable increase in the total number of AcChoRs on the cell surface measured with a labeled receptor probe. The results imply that the neurons have functional and nonfunctional pools of AcChoRs and that functional receptors can be recruited from intracellular receptors or from nonfunctional receptors on the cell surface by a cAMP-dependent process. A cAMP-dependent regulation of the number of functional neurotransmitter receptors would provide a reversible mechanism by which cell-cell interactions could modulate synaptic transmission in the nervous system.

The properties of acetylcholine receptor (AChR) channels on chick ciliary ganglion neurons in culture were examined using patch-clamp recording techniques. Acetylcholine (ACh) was applied by rapid microperfusion. Whole-cell current noise analysis revealed a single class of functional receptors on the neurons. Dose-response studies indicated a Kd of about 36 microM and a Hill coefficient of 1.5-1.7, predicting 2 ACh binding sites per receptor. Both fast and slow components of receptor desensitization were observed. Single-channel recordings from excised outside-out patches of soma membrane exposed to 2-5 microM ACh indicated a single-channel conductance of 40 pS, a reversal potential of -9 mV, a mean open duration of 1 msec, and an opening probability of 0.34. The kinetic behavior of the channels was provisionally described by a 3-closed, 1-open state model for receptor activation. In all of these properties, AChRs of ciliary ganglion neurons resemble those on skeletal muscle fibers. Growing the neurons in an elevated K+ concentration produced a 2-3-fold decrease in peak whole-cell currents induced by ACh under standard test conditions, without altering any of the single-channel properties described above. Neither changes in cholinesterase activity nor receptor distribution accounted for the decrease. Instead, calculations indicated that elevated K+ reduced the ACh response by decreasing the number of functional AChRs on the neurons. No K+-dependent decrease is observed, however, in the number of total receptors on the neurons detected either by a monoclonal antibody specific for the receptor or by an alpha-neurotoxin that binds to the receptor and blocks its function. Moreover, the number of receptors detected by the 2 probes is at least 10-fold greater than the calculated number of functional receptors. The findings suggest that only a small fraction of the AChRs on the neuronal surface is functional and that the cell can alter the ratio of functional and nonfunctional receptors in response to growth conditions.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widespread, diverse ion channels involved in synaptic signaling, addiction, and disease. Despite their importance, the relationship between native nAChR subunit composition and function remains poorly defined. Chick ciliary ganglion neurons express two major nAChR types: those recognized by ␣-bungarotoxin (␣Bgt), nearly all of which contain only ␣7 subunits (␣7-nAChRs) and those insensitive to ␣Bgt, which contain ␣3, ␣5, ␤4, and, in some cases, ␤2 subunits (␣3*-nAChRs). We explored the relationship between nAChR composition and channel function using toxins recognizing ␣7 subunits (␣Bgt), and ␣3/␤4 (␣-conotoxin-AuIB), or ␣3/␤2 (␣-conotoxin-MII) subunit interfaces to perturb responses induced by nicotine, ␣7-, or ␣3-selective agonists (GTS-21 or epibatidine, respectively). Using these reagents, fast-decaying whole-cell current components were attributed solely to ␣7-nAChRs, and slow-decaying components mostly to ␣3*-nAChRs. In outside-out patches, nicotine activated brief 60-and 80-pS single nAChR channel events, and mixed-duration 25-and 40-pS nAChR events. Subsequently, 60-and 80-pS nAChR events and most brief 25-and 40-pS events were attributed to ␣7-nAChRs, and long 25-and 40-pS events to ␣3*-nAChRs. ␣3*-nAChRs lacking ␤2 subunits seemed responsible for long 25 pS nAChR events, whereas those containing ␤2 subunits mediated the long 40 pS nAChR events that dominate single-channel records. These results reveal greater functional heterogeneity for ␣7-nAChRs than previously expected and indicate that ␤2 subunits contribute importantly to ␣3*-nAChR function. By linking structural to functional nAChR subtypes, the findings also illustrate a useful pharmacological strategy for selectively targeting nAChRs.