Pediatric Oral Motor Feeding Assessments: A Systematic Review.

AIM: To describe the clinical properties and psychometric soundness of pediatric oral motor feeding assessments. METHODS: A systematic search was conducted using Medline, CINAHL, EMBASE, PsycInfo, and HAPI databases. Assessments were analyzed for their clinical and psychometric characteristics. RESULTS: 12 assessment tools were identified to meet the inclusion/exclusion criteria. Clinical properties varied from assessments evaluating oral-motor deficits, screening to identify feeding problems, and monitoring feeding progress. Most assessments were designed for children with developmental disabilities or cerebral palsy. Eleven assessments had psychometric evidence, of these nine had reliability and validity testing (Ability for Basic Feeding and Swallowing Scale for Children, Behavioral Assessment Scale of Oral Functions in Feeding, Dysphagia Disorder Survey, Functional Feeding Assessment-modified, Gisel Video Assessment, Montreal Children's Hospital Feeding Scale, Oral Motor Assessment Scale, Schedule for Oral Motor Assessment, and Screening Tool of Feeding Problems Applied to Children). The Brief Assessment of Motor Function-Oral Motor Deglutition and the Pediatric Assessment Scale for Severe Feeding Problems had reliability testing only. The Slurp Test was not tested for any psychometric properties. Overall, psychometric evidence was inconsistent and inadequate for the evaluative tools.

A self-paced oral feeding system that enhances preterm infants' oral feeding skills.

AIM: Very low birth weight (VLBW) infants have difficulty transitioning to independent oral feeding, be they breast- or bottle-feeding. We developed a 'self-paced' feeding system that eliminates the natural presence of the positive hydrostatic pressure and internal vacuum build-up within a bottle during feeding. Such system enhanced these infants' oral feeding performance as monitored by overall transfer (OT; % ml taken/ml prescribed), rate of transfer (RT; ml/min over an entire feeding). This study hypothesizes that the improvements observed in these infants resulted from their ability to use more mature oral feeding skills (OFS). METHODS: 'Feeders and growers' born between 26-29 weeks gestation were assigned to a control or experimental group fed with a standard or self-paced bottle, respectively. They were monitored when taking 1-2 and 6-8 oral feedings/day. OFS was monitored using our recently published non-invasive assessment scale that identifies 4 maturity levels based on infants' RT and proficiency (PRO; % ml taken during the first 5 min of a feeding/total ml prescribed) during bottle feeding. RESULTS: Infants oral feeding outcomes, i.e., OT, RT, PRO, and OFS maturity levels were enhanced in infants fed with the self-paced vs. standard bottle (p ≤ 0.007). CONCLUSION: The improved oral feeding performance of VLBW infants correlated with enhanced OFS. This study is a first to recognize that VLBW infants' true OFS are more mature than recognized. We speculate that the physical properties inherent to standard bottles that are eliminated with the self-paced system interfere with the display of their true oral feeding potential thereby hindering their overall oral feeding performance.

Effect of an oral stimulation program on sucking skill maturation of preterm infants.

This study assessed the effect of an oral stimulation program on the maturation of sucking skills of preterm infants. Thirty-two preterm infants (13 males, 19 females), appropriate size for gestational age (gestational age at birth 28 wks, SD 1.2wks; birthweight 1002g, SD 251g), were randomly placed into experimental and control groups. The experimental group received a daily 15-minute oral stimulation program, consisting of stroking the peri- and intra-oral structures, for 10 days before the start of oral feedings. Sucking measures were monitored with a specially-designed nipple-bottle apparatus. Results indicate that the experimental group achieved full oral feedings 7 days sooner than the control group, and demonstrated greater overall intake (%), rate of milk transfer (mL/min), and amplitude of the expression component of sucking (mmHg). There was no difference in sucking stage maturation, sucking frequency, and amplitude of the suction component of sucking. Endurance, defined as ability to sustain the same sucking stage, sucking burst duration, and suction and expression amplitudes throughout a feeding session, was not significantly different between the two groups. The stimulation program enhanced the expression component of sucking, resulting in better oral feeding performance.

Nicotinic cholinergic stimulation promotes survival and reduces motility of cultured rat cerebellar granule cells.

Despite many studies on the functional expression of neuronal nicotinic acetylcholine receptors (nAChRs), an exhaustive description of the long-term effects of nicotine (Nic) stimulation in cerebellar granules is still far to be completed. For this reason, we addressed the experiments stimulating cultured cerebellar granule neurons (CGN) with Nic, focusing on the effects on cell motility and survival. Using electrophysiological and Ca(2+)-fluorescence techniques, we found a subset of rat CGN that responded to Nic by inward whole cell currents and by short-delay Ca(2+) transients. These responses were mediated through both homomeric and heteromeric nAChRs, as assessed by their sensitivity to alpha-bungarotoxin (alpha-BTX), dihydro-beta-erythroidine (DHbetaE), methyllicaconitine (MLA) and 5-hydroxyindole (5OH-indole). Once established the expression of alpha-BTX-sensitive and insensitive nAChRs and their ability to trigger Ca(2+) responses in CGN, we aimed at investigating their possible role on cell survival and motility. We demonstrate that Nic stimulation significantly increases the survival of CGN exposed to the apoptosis-promoting low K(+) medium. This anti-apoptotic effect is likely mediated through alpha7* nAChRs since we found that it was mimicked by choline, was insensitive to DHbetaE and was fully inhibited by alpha-BTX. Furthermore, we report that Nic negatively modulates CGN motility, reducing the basal cell movement through a pored membrane by the activation of alpha-BTX-insensitive nAChRs. We conclude that CGN express various types of nAChRs, which are differently involved in regulating Nic-mediated modulation of cell survival and migration, and we suggest potential regulatory roles for cholinergic receptors during cerebellar development.

Ca(2+) permeability of human heteromeric nAChRs expressed by transfection in human cells.

The Ca(2+) permeability of the human heteromeric alpha 3 beta 4, alpha 4 beta 2 and alpha 4 beta 4 neuronal nicotinic acetylcholine receptors (nAChRs) was estimated by measuring the fractional Ca(2+) current (P(f)) flowing through the ligand-activated receptor-channels. Simultaneous recordings of transmembrane currents and fluorescence transients, using the whole-cell patch-clamp technique combined with fura-2 fluorescence microscopy, were performed in transiently transfected human cells. The human alpha 4 beta 2 nAChR showed a P(f) value of 2.6%, while the human alpha 3 beta 4 nAChR showed a similar P(f) value of 2.7%. Conversely, alpha 4 beta 4 nAChR exhibited a P(f) value (1.5%) significantly smaller than those of both alpha 4 beta 2 and alpha 3 beta 4 nAChRs. In test experiments performed in HEK 293 cells stably expressing rat GluR1 AMPA receptor subunit, we repeated the determination of P(f), whose value (3.2%) has previously been reported by others using the same fluorescent dye; and we found a very similar P(f) value (3.5%). In further test experiments, we found that P(f) values of chick alpha 3 beta 4 (4.4%) and alpha 4 beta 4 (2.1%) matched those previously reported by us using confocal fluorescence microscopy. Thus, our findings are consistent with those elsewhere reported even using different experimental procedures, giving a strong support to the following sequence of Ca(2+) permeability: h-alpha 3 beta 4>h-alpha 4 beta 2>h-alpha 4 beta 4.

Chemokine receptor CXCR2 regulates the functional properties of AMPA-type glutamate receptor GluR1 in HEK cells.

Experiments were conducted in both HEK cells and cerebellar neurons to investigate whether CXC chemokine receptor 2 (CXCR2) is functionally coupled to GluR1. The co-expression of CXCR2 with GluR1 in HEK cells increased (i) the GluR1 "apparent" affinity for the transmitter; (ii) the GluR1 channel open probability; and (iii) GluR1 binding site cooperativity upon CXCR2 stimulation with CXC chemokine ligand 2 (CXCL2). The affinity of C-terminal-deleted GluR1 for glutamate (Glu) remained stable instead. Furthermore, CXCL2 increased the binding site cooperativity of AMPA receptors in rat cerebellar granule cells; and the amplitude of spontaneous excitatory postsynaptic current (sEPSCs) in Purkinje neurons (PNs). Our findings indicate that the coupling of CXCR2 with GluR1 may modulate glutamatergic synaptic transmission.

Serotonin antagonizes the human neuronal alpha7 nicotinic acetylcholine receptor and becomes an agonist after L248T alpha7 mutation.

The effects of serotonin (5-hydroxytryptamine or 5HT) on chick alpha7 nicotinic receptors have already been described. However similar studies on human alpha7 receptors have been lacking. To begin to fill this deficiency, studies were made on wild-type and mutant human alpha7 (halpha7) receptors expressed in Xenopus oocytes or human BOSC 23 cells. In oocytes wild-type halpha7 receptors were blocked by 5HT, and this block was voltage-dependent. In contrast, 5HT acted as an agonist on halpha7-mutant receptors (L248T). Outside-out membrane-patches from BOSC 23 cells expressing halpha7-mutant receptors exhibited spontaneous channel openings of two conductance levels (59 pS and 76 pS) and short mean open time (0.9 ms). halpha7-Mutant channels activated by nicotine or 5HT displayed similar conductances and high Ca(2+) permeability; but longer duration (2.7 ms) than the spontaneous openings. Mutations at Cys190 and Cys191, in the extracellular N-terminus of the human alpha7 gene, did not prevent receptor expression and incorporation in the oocyte membrane (determined by alpha-bungarotoxin binding). However, both 5HT and nicotine were incapable of gating the channels, indicating that the mutated Cys residues are in, or near, the 5HT- and nicotine-binding site. This is the first report that alpha7 receptors have spontaneous openings; and that 5HT is an agonist of halpha7-mutant receptors, and an antagonist of halpha7-wild-type receptors, through interactions at, or near the acetylcholine-binding sites.

Human neuronal threonine-for-leucine-248 alpha 7 mutant nicotinic acetylcholine receptors are highly Ca2+ permeable.

A cDNA coding for the human neuronal nicotinic alpha7 receptor subunit with Leu-248 mutated to threonine was expressed in Xenopus oocytes. When activated by acetylcholine (AcCho), the receptors expressed generated currents that had low desensitization, linear current-voltage relation, and high apparent affinity for both AcCho and nicotine. These characteristics are similar to those already described for the chick threonine-for-leucine-247 alpha7 nicotinic AcCho receptor (nAcChoR) mutant (L247Talpha7). These properties were all substantially maintained when the human L248Talpha7 mutant was transiently expressed in human Bosc 23 cells. Simultaneous whole-cell clamp and fluorescence measurements with the Ca(2+) indicator dye Fura-2 showed that nicotine induced a Ca(2+) influx in standard 2 mM Ca(2+) solution. The average fractional Ca(2+) current flowing through L248Talpha7 nAcChoRs was 6.7%, which is larger than that flowing through muscle alpha(beta)epsilon(delta) nAcChoRs (4.1%). The relative Ca(2+) permeability, determined in oocytes in the absence of Cl(-), was measured from the shift in reversal potential caused by increasing the external Ca(2+) concentration from 1 to 10 mM. The human wild-type alpha7 nAcChoR was found to be more permeable than the L248Talpha7 mutant to Ca(2+). Our findings indicate that the Ca(2+) permeability of the homomeric alpha7 nAcChoR is larger than that of the heteromeric neuronal nicotinic receptors studied to date and is possibly similar to that of the N-methyl-D-aspartate subtype of brain glutamate receptors.

Fast potentiation of glycine receptor channels of intracellular calcium in neurons and transfected cells.

Inhibitory glycine receptors (GlyRs) are mainly expressed in the spinal cord and in the midbrain, where they control motor and sensory pathways. We describe here a fast potentiation of GlyR by intracellular Ca2+. This phenomenon was observed in rat spinal cord neurons and in transfected human cell lines. Potentiation develops in <100 ms, is proportional to Ca2+ influx, and is characterized by an increase in GlyR apparent affinity for glycine. Phosphorylation and G protein pathways appear not to be involved in the potentiation mechanism. Single-channel recordings in cell-attached and excised patches, as well as whole-cell data suggest the presence of a diffusible cytoplasmic factor that modulates the GlyR channel gating properties. Ca2+-induced potentiation may be important for rapid modulation of glycinergic synapses.

Strychnine activates neuronal alpha7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains.

Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric alpha7 receptors, and that mutating Leu-247 of the alpha7 nicotinic AcCho receptor-channel domain (L247Talpha7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type alpha7 or L247Talpha7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Talpha(7) receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189-190Salpha7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker alpha-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic alpha7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical alpha7 subunits may be sufficient to determine the functional properties of alpha7 receptors.

Functional integrity of green fluorescent protein conjugated glycine receptor channels.

The alpha subunit (alphaZ1) of the zebrafish glycine receptor (GlyR) has been N-terminus fused with green fluorescent protein (GFP). We found that both pharmacological and electrophysiological properties of this chimeric alphaZ1-GFP are indistinguishable from those of the wild-type receptor when expressed in Xenopus oocytes and cell lines. The apparent affinities of this receptor for agonists (glycine, taurine and GABA), and the antagonist (strychnine) are unchanged, and single channel kinetics are not altered. In the same expression systems, alphaZ1-GFP was visualized using fluorescence microscopy. Fluorescence was distributed anisotropically across cellular membranes. In addition to the Golgi apparatus and endoplasmic reticulum, its presence was also detected on the plasmalemma, localized at discrete hot-spots which were identified as sites of high membrane turnover. Overall, the preservation in alphaZ1-GFPs of the wild type receptor functional properties makes it a promising new tool for further in situ investigations of GlyR expression, distribution and function.

Comparison of glycine and GABA actions on the zebrafish homomeric glycine receptor.

1. Glycine and GABA can be co-released from the same presynaptic terminals and in lower vertebrates they can activate the same glycine receptors (GlyRs). Thus we examined the effects of these two inhibitory transmitters on the homomeric GlyRs formed by the alphaZ1 subunit, of the zebrafish using two expression systems: Xenopus oocytes and the human BOSC 23 cell line. 2. The apparent affinity (EC50) of alphaZ1 for these neurotransmitters was highly variable. In Xenopus oocytes the EC50 ranged from 37 to 360 microM (mean +/- s. d. EC50 116 +/- 75 microM, n = 83) for glycine and from 8 to 120 mM (mean EC50 40 +/- 30 mM, n = 37) for GABA. 3. In BOSC cells the EC50 varied from 9 to 92 microM (mean EC50 33 +/- 17 microM, n = 19) and from 0.7 to 19.1 mM (mean EC50 4.9 +/- 4.7 mM, n = 29) for glycine and GABA, respectively. 4. GABA activated alphaZ1 GlyRs either as a weak or full agonist: its efficacy (defined as Imax,GABA/Imax,Gly) was related to EC50 by an exponential relationship. A linear relationship was observed between EC50 values for GABA and glycine. 5. In outside-out patches, GABA and glycine activated alphaZ1 with identical single-channel conductances (85-100 pS), but with different kinetics and marked effect of concentration on burst duration for glycine only. 6. In outside-out patches deactivation time constants were concentration dependent for glycine, but not for GABA. 7. Our data demonstrate that the kinetics of glycine and GABA interactions with alphaZ1 are different and that they determine the properties of these neurotransmitter actions on the GlyR.

Cloning, expression and electrophysiological characterization of glycine receptor alpha subunit from zebrafish.

The glycine receptor is a ligand-gated anion channel protein, providing inhibitory drive within the nervous system. We report here the isolation and functional characterization of a novel alpha subunit (alphaZ1) of the glycine receptor from adult zebrafish (Danio rerio) brain. The predicted amino acid sequence is 86%, 81% and 77% identical to mammalian isoforms alpha1, alpha3 and alpha2, respectively. AlphaZ1 exhibits many of the molecular features of mammalian alpha1, but the sequence patterns in the M4 and C-terminal domains are more similar to alpha2/alpha3. Phylogenetic analysis indicates that alphaZ1 is more closely related to the mammalian alpha1 subunits, being positioned, however, on a distinct branch. The alphaZ1 messenger RNA is 9.5 kb, similar to that described previously for alpha1 messenger RNAs. When expressed in Xenopus oocytes or a human cell line (BOSC 23), alphaZ1 forms a homomeric receptor which is activated by glycine and antagonized by strychnine. This receptor demonstrates unexpectedly high sensitivity to taurine and can also be activated by GABA. These results are consistent with physiological findings in lamprey and goldfish, and they suggest that this teleost fish glycine receptor displays a lower selectivity to neurotransmitters than that reported for glycine mammalian receptors.

The neuronal alpha6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells.

We examine some of the biological and physiological properties of the avian alpha6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, alpha6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as alpha3, beta2 and beta4. In contrast, alpha6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the alpha6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick alpha6 in combination with other chick nAChR neuronal subunits. Heterologously expressed alpha6 and beta4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 microM). Likewise, ACh elicits measurable currents in cells transfected with alpha6 and beta2. Hill analysis of the dose-response curves in cells transfected with alpha3, beta4 and alpha6 cDNAs, suggests the assembly of functional alpha3beta4alpha6 receptor, with an apparent affinity for ACh threefold lower than alpha3beta4. Our results indicate that alpha6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.

Functional oral-motor skills: Do they change with age?

Dysphagia, a difficulty eating or drinking, appears to increase with age and is a concern for our growing elderly population. Mastication, tongue mobility, and lip closure are skills of the oral phase of ingestion, and have been shown to deteriorate with age. However, it is not clear whether these changes affect functional feeding. It is also unclear whether dysphagia is the result of the aging process itself, or whether it is secondary to disease. Therefore, the purpose of this study was to identify changes during the oral phase of ingestion in a group of healthy seniors. Functional feeding skills and oral praxis abilities were measured in 79 healthy adults aged 60-97 years. The Modified Functional Feeding Assessment (FFAm) subscale of the Multidisciplinary Feeding Profile (MFP) and the Oral Praxis Subtest (OPS) of the Southern California Sensory Integration Test were administered respectively. An interview followed to obtain information on denture wear, use of hearing aids and glasses, and types of foods avoided. Seniors maintained functional feeding skills throughout the four decades studied. These skills were not age-dependent, but depended on whether or not subjects wore full dentures. Even though all of the seniors maintained functional feeding skills, more seniors in the younger group (7th decade 60%, 8th decade 67%) had difficulty with a variety of food textures such as soft, hard, fibrous, and some with tough skins, than the older group (9th decade 40%, 10th decade 44%). Oral praxis abilities were correlated significantly with age, but not with hearing aid use. Overall, healthy seniors maintained their functional feeding and oral praxis skills. Good health and natural dentition appear to be excellent indicators for functional feeding ability.

Ca2+ permeability of mouse and chick nicotinic acetylcholine receptors expressed in transiently transfected human cells.

1. Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane currents and fluorescence transients using the whole-cell patch-clamp technique, and confocal microscopy with the Ca2+ indicator dye fluo-3. 2. The fractional Ca2+ current, Pf, of nAChRs was evaluated as the normalized ratio of nicotine-evoked fluorescence transient over total charge entering the cell (F/Q ratio). Mouse fetal muscle nAChR channels had a Pf, alphabetagammadelta value of 2.1 %. The substitution of the gamma subunit with the epsilon subunit resulted in a 2-fold increase in Pf (4.2 %). The difference in Ca2+ permeability was confirmed by determination of Ca2+/Cs+ permeability ratios. 3. Among the chick neuronal nAChRs tested, Pf,alpha3beta4 was 4.6 %, while Pf, alpha4beta4 and Pf,alpha4beta2 were 3.0 % and 2.9 %, respectively. 4. The amplitude of the current elicited by the activation of alpha3beta4 nAChRs increased as the external Ca2+ concentration was raised from 2 to 110 mM, whereas currents flowing through all other nAChRs tested were reduced to various extents. 5. Our findings indicate that the adult-type muscle nAChR (alphabetaepsilondelta) is more permeable to Ca2+ than the fetal-type (alphabetagammadelta), while ganglionic-like alpha3beta4 nAChR is more permeable to Ca2+ than the examined alpha4-containing nAChRs. The functional significance is discussed.

Alpha 5 subunit forms functional alpha 3 beta 4 alpha 5 nAChRs in transfected human cells.

nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.

Functional properties of neuronal nicotinic acetylcholine receptor channels expressed in transfected human cells.

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat alpha 3 beta 4, chick alpha 3 beta 2, alpha 4 beta 2, alpha 7 and alpha 8), expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of alpha 8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19-23 and 32-45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric alpha 7 and alpha 8 nAChRs (6-7 ms) than for heteromeric nAChRs (1-3 ms), with the exception of alpha 3 beta 4 nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (alpha 3 beta 4 and alpha 3 beta 2) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChR channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.

Cholinergic stimulation of human microcytoma cell line H69.

Experiments were addressed to investigate the mechanisms by which cholinergic stimulation is coupled to the enhancement of proliferation of small cell lung cancer cells H69. Muscarinic stimulation triggers the release of cytosolic Ca2+ and of inositol(1,4,5)trisphosphate with comparable time courses. The presence of alpha-bungarotoxin or the absence of Ca2+ in external medium suppresses enhancement of clonal growth induced by brief applications of nicotine. Here we suggest that Ca2+ mobilization represents a trigger for the enhancement of small cell lung cancer cell proliferation upon cholinergic stimulation.

Identification of a determinant of acetylcholine receptor gating kinetics in the extracellular portion of the gamma subunit.

A large body of structure-function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR gamma subunit, which lacks the 52 amino acids within the extracellular amino-terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short gamma subunit [gamma(s)] with alpha, beta and delta subunits in human cells and recorded single-channel currents from the resulting AChRs. Our findings show that replacement of the gamma by the gamma(s) subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that alpha beta gamma(s) delta AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the membrane potential is hyperpolarized. Together, these findings indicate that the domain in the extracellular amino-terminal half of the gamma subunit that encompasses a conserved disulphide loop and a critical tyrosine residue implicated in receptor oligomerization and insertion at the cell surface is a functional motif that also modulates AChR channel gating kinetics. The results also provide a molecular explanation of the functional diversity exhibited by skeletal muscle AChRs during development.