Dynamic Heterogeneity Shapes Patterns of Spiral Ganglion Activity.

Neural response properties that typify primary sensory afferents are critical to fully appreciate because they establish and, ultimately represent, the fundamental coding design used for higher-level processing. Studies illuminating the center-surround receptive fields of retinal ganglion cells, for example, were ground-breaking because they determined the foundation of visual form detection. For the auditory system, a basic organizing principle of the spiral ganglion afferents is their extensive electrophysiological heterogeneity establishing diverse intrinsic firing properties in neurons throughout the spiral ganglion. Moreover, these neurons display an impressively large array of neurotransmitter receptor types that are responsive to efferent feedback. Thus, electrophysiological diversity and its neuromodulation are a fundamental encoding mechanism contributed by the primary afferents in the auditory system. To place these features into context, we evaluated the effects of hyperpolarization and cAMP on threshold level as indicators of overall afferent responsiveness in CBA/CaJ mice of either sex. Hyperpolarization modified threshold gradients such that distinct voltage protocols could shift the relationship between sensitivity and stimulus input to reshape resolution. This resulted in an "accordion effect" that appeared to stretch, compress, or maintain responsivity across the gradient of afferent thresholds. cAMP targeted threshold and kinetic shifts to rapidly adapting neurons, thus revealing multiple cochleotopic properties that could potentially be independently regulated. These examples of dynamic heterogeneity in primary auditory afferents not only have the capacity to shift the range, sensitivity, and resolution, but to do so in a coordinated manner that appears to orchestrate changes with a seemingly unlimited repertoire.SIGNIFICANCE STATEMENT How do we discriminate the more nuanced qualities of the sound around us? Beyond the basics of pitch and loudness, aspects, such as pattern, distance, velocity, and location, are all attributes that must be used to encode acoustic sensations effectively. While higher-level processing is required for perception, it would not be unexpected if the primary auditory afferents optimized receptor input to expedite neural encoding. The findings reported herein are consistent with this design. Neuromodulation compressed, expanded, shifted, or realigned intrinsic electrophysiological heterogeneity to alter neuronal responses selectively and dynamically. This suggests that diverse spiral ganglion phenotypes provide a rich substrate to support an almost limitless array of coding strategies within the first neural element of the auditory pathway.

Amplification of input differences by dynamic heterogeneity in the spiral ganglion.

A defining feature of type I primary auditory afferents that compose ∼95% of the spiral ganglion is their intrinsic electrophysiological heterogeneity. This diversity is evident both between and within unitary, rapid, and slow adaptation (UA, RA, and SA) classes indicative of specializations designed to shape sensory receptor input. But to what end? Our initial impulse is to expect the opposite: that auditory afferents fire uniformly to represent acoustic stimuli with accuracy and high fidelity. Yet this is clearly not the case. One explanation for this neural signaling strategy is to coordinate a system in which differences between input stimuli are amplified. If this is correct, then stimulus disparity enhancements within the primary afferents should be transmitted seamlessly into auditory processing pathways that utilize population coding for difference detection. Using sound localization as an example, one would expect to observe separately regulated differences in intensity level compared with timing or spectral cues within a graded tonotopic distribution. This possibility was evaluated by examining the neuromodulatory effects of cAMP on immature neurons with high excitability and slow membrane kinetics. We found that electrophysiological correlates of intensity and timing were indeed independently regulated and tonotopically distributed, depending on intracellular cAMP signaling level. These observations, therefore, are indicative of a system in which differences between signaling elements of individual stimulus attributes are systematically amplified according to auditory processing constraints. Thus, dynamic heterogeneity mediated by cAMP in the spiral ganglion has the potential to enhance the representations of stimulus input disparities transmitted into higher level difference detection circuitry.NEW & NOTEWORTHY Can changes in intracellular second messenger signaling within primary auditory afferents shift our perception of sound? Results presented herein lead to this conclusion. We found that intracellular cAMP signaling level systematically altered the kinetics and excitability of primary auditory afferents, exemplifying how dynamic heterogeneity can enhance differences between electrophysiological correlates of timing and intensity.

ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity.

Ataxia telangiectasia is a multisystemic disorder that includes a devastating neurodegeneration phenotype. The ATM (ataxia-telangiectasia mutated) protein is well-known for its role in the DNA damage response, yet ATM is also found in association with cytoplasmic vesicular structures: endosomes and lysosomes, as well as neuronal synaptic vesicles. In keeping with this latter association, electrical stimulation of the Schaffer collateral pathway in hippocampal slices from ATM-deficient mice does not elicit normal long-term potentiation (LTP). The current study was undertaken to assess the nature of this deficit. Theta burst-induced LTP was reduced in Atm(-/-) animals, with the reduction most pronounced at burst stimuli that included 6 or greater trains. To assess whether the deficit was associated with a pre- or postsynaptic failure, we analyzed paired-pulse facilitation and found that it too was significantly reduced in Atm(-/-) mice. This indicates a deficit in presynaptic function. As further evidence that these synaptic effects of ATM deficiency were presynaptic, we used stochastic optical reconstruction microscopy. Three-dimensional reconstruction revealed that ATM is significantly more closely associated with Piccolo (a presynaptic marker) than with Homer1 (a postsynaptic marker). These results underline how, in addition to its nuclear functions, ATM plays an important functional role in the neuronal synapse where it participates in the regulation of presynaptic vesicle physiology.

The BDNF Val66Met polymorphism impairs NMDA receptor-dependent synaptic plasticity in the hippocampus.

The Val66Met polymorphism in the brain-derived neurotrophic factor (BDNF) gene results in a defect in regulated release of BDNF and affects episodic memory and affective behaviors. However, the precise role of the BDNF Val66Met polymorphism in hippocampal synaptic transmission and plasticity has not yet been studied. Therefore, we examined synaptic properties in the hippocampal CA3-CA1 synapses of BDNF(Met/Met) mice and matched wild-type mice. Although basal glutamatergic neurotransmission was normal, both young and adult mice showed a significant reduction in NMDA receptor-dependent long-term potentiation. We also found that NMDA receptor-dependent long-term depression was decreased in BDNF(Met/Met) mice. However, mGluR-dependent long-term depression was not affected by the BDNF Val66Met polymorphism. Consistent with the NMDA receptor-dependent synaptic plasticity impairment, we observed a significant decrease in NMDA receptor neurotransmission in the CA1 pyramidal neurons of BDNF(Met/Met) mice. Thus, these results show that the BDNF Val66Met polymorphism has a direct effect on NMDA receptor transmission, which may account for changes in synaptic plasticity in the hippocampus.

EphA activation overrides the presynaptic actions of BDNF.

The adult pattern of neural connectivity is shaped by repulsive and attractive factors, many of which are modulated by activity. Although much is known about the actions of these factors when studied in isolation, little is known about how they interact. To address this question, we examined the effects of sequential or coapplication of brain-derived neurotrophic factor (BDNF) and Fc-conjugated ephrin-A5 or EphA5 in cultured embryonic hippocampal neurons. BDNF promotes neurite outgrowth and synapse formation, and when applied acutely, it elicits an increase in ongoing synaptic activity. Members of the ephrin family of ligands and receptors can be repulsive and prevent formation of synaptic contacts. Acute exposure to either ephrin-A5-Fc or EphA5-Fc transiently enhanced synaptic activity when applied alone, but when applied prior to BDNF, they dramatically reduced the electrophysiological effects of the neurotrophin. Conversely, BDNF had no effect on subsequently applied ephrin-A5-Fc or EphA5-Fc. Consistent with this, ephrin-A5-Fc also prevented BDNF-induced activation of p42/44 MAPK. The effect of ephrin-A5-Fc appears to be presynaptic, as it prevented the BDNF-induced increase in spontaneous miniature postsynaptic current frequency, whereas EphA5-Fc did not. These results suggest that these factors can be categorized differently, with the contact-mediated activation of EphA receptors by ephrin-A5 overriding the diffusion-mediated effect of BDNF.

The glial or neuronal fate choice of oligodendrocyte progenitors is modulated by their ability to acquire an epigenetic memory.

The identity of any cell type is determined by the specific pattern of gene expression. We show here that the ability of oligodendrocyte progenitors to acquire the identity of myelin-expressing cells or choose alternative fates is dependent on the activity of histone deacetylases. Using gene expression profiling, electrophysiological recordings, transplantation studies, and pharmacological inhibition, we demonstrate that specified NG2+ oligodendrocyte progenitors are plastic cells, whose decision to initiate an oligodendrocytic rather than astrocytic or neuronal program of gene expression requires the establishment of an epigenetic identity that is initiated by histone deacetylation.

Single-cell characterization of retrograde signaling by brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a key regulator of hippocampal synaptic plasticity in the developing and adult nervous system. It can be released from pyramidal neuron dendrites in an activity-dependent manner and has therefore been suggested to serve as a signal that provides the retrograde intercellular communication necessary for Hebbian plasticity and hippocampal-dependent learning. Although much has been learned about BDNF function by field stimulation of hippocampal neurons, it is not known whether moderate action potential-independent depolarization of single cells is capable of releasing sufficient BDNF to influence transmission at individual synapses. In this study, we show directly at the single-cell level that such modulation can occur. By using K-252a, anti-BDNF antibody, and interruption of regulated release, we confirm a model in which postsynaptic depolarization elicits calcium-dependent release of BDNF that diffuses retrogradely and enhances presynaptic transmitter release.

Early presynaptic and late postsynaptic components contribute independently to brain-derived neurotrophic factor-induced synaptic plasticity.

Trophin-induced synaptic plasticity consists of both presynaptic and postsynaptic processes. The potential interdependence of these mechanisms and their temporal relationships are undefined. The synaptic vesicle protein Rab3A is required for the early, initial 10 min phase but not for the later phase of BDNF-enhanced transmission. We now examine the temporal distinction and mechanistic relationships between these phases of BDNF action. Rab3A mutant cells did not exhibit increased miniature EPSC frequency in response to BDNF in cell culture, indicating an absence of the presynaptic component. In contrast, BDNF enhanced postsynaptic glutamate-induced current in the mutant neurons as in the wild type, indicating that the postsynaptic component of the response was intact. Finally, the postsynaptic NMDA receptor subunit NR2B was phosphorylated at Tyr1472 by BDNF in Rab3A knock-outs, as shown previously in wild type. Our results are the first to demonstrate that presynaptic and postsynaptic components of BDNF-enhanced synaptic activity are independent and temporally distinct.

Brain-derived neurotrophic factor rapidly increases NMDA receptor channel activity through Fyn-mediated phosphorylation.

Brain-derived neurotrophic factor (BDNF) is a potent modulator of hippocampal synaptic plasticity. Previously, we found that one of the targets of BDNF modulation is NR2B-containing NMDA receptors. Furthermore, exposure to the trophin rapidly increases NMDA receptor activity and enhances tyrosine phosphorylation of NR2B in cortical and hippocampal postsynaptic densities (PSDs), potentially linking receptor phosphorylation to synaptic plasticity. To define the specific NR2B residue(s) regulated by BDNF, we focused on tyrosine 1472, phosphorylation of which increases after LTP. BDNF rapidly increased phosphorylation in cortical PSDs. The tyrosine kinase Fyn is critical since BDNF-dependent phosphorylation was abolished in Fyn knockout mice. Single-channel patch clamp recordings showed that Fyn is required for the increase in NMDA receptor activity elicited by BDNF. Collectively, our results suggest that BDNF enhances phosphorylation of NR2B tyrosine 1472 through activation of Fyn, leading to alteration of NMDA receptor activity and increased synaptic transmission.

The impact of glutamine supplementation on the symptoms of ataxia-telangiectasia: a preclinical assessment.

BACKGROUND: Our previous studies of Alzheimer's disease (AD) suggested that glutamine broadly improves cellular readiness to respond to stress and acts as a neuroprotectant both in vitro and in AD mouse models. We now expand our studies to a second neurodegenerative disease, ataxia-telangiectasia (A-T). Unlike AD, where clinically significant cognitive decline does not typically occur before age 65, A-T symptoms appear in early childhood and are caused exclusively by mutations in the ATM (A-T mutated) gene. RESULTS: Genetically ATM-deficient mice and wild type littermates were maintained with or without 4 % glutamine in their drinking water for several weeks. In ATM mutants, glutamine supplementation restored serum glutamine and glucose levels and reduced body weight loss. Lost neurophysiological function assessed through the magnitude of hippocampal long term potentiation was significantly restored. Glutamine supplemented mice also showed reduced thymus pathology and, remarkably, a full one-third extension of lifespan. In vitro assays revealed that ATM-deficient cells are more sensitive to glutamine deprivation, while supra-molar glutamine (8 mM) partially rescued the reduction of BDNF expression and HDAC4 nuclear translocation of genetically mutant Atm(-/-) neurons. Analysis of microarray data suggested that glutamine metabolism is significantly altered in human A-T brains as well. CONCLUSION: Glutamine is a powerful part of an organism's internal environment. Changes in its concentrations can have a huge impact on the function of all organ systems, especially the brain. Glutamine supplementation thus bears consideration as a therapeutic strategy for the treatment of human A-T and perhaps other neurodegenerative diseases.

Brain-derived neurotrophic factor-induced gene expression reveals novel actions of VGF in hippocampal synaptic plasticity.

Synaptic strengthening induced by brain-derived neurotrophic factor (BDNF) is associated with learning and is coupled to transcriptional activation. However, identification of the spectrum of genes associated with BDNF-induced synaptic plasticity and the correlation of expression with learning paradigms in vivo has not yet been studied. Transcriptional analysis of BDNF-induced synaptic strengthening in cultured hippocampal neurons revealed increased expression of the immediate early genes (IEGs), c-fos, early growth response gene 1 (EGR1), activity-regulated cytoskeletal-associated protein (Arc) at 20 min, and the secreted peptide VGF (non-acronymic) protein precursor at 3 hr. The induced genes served as prototypes to decipher mechanisms of both BDNF-induced transcription and plasticity. BDNF-mediated gene expression was tyrosine kinase B and mitogen-activated protein kinase-dependent, as demonstrated by pharmacological studies. Single-cell transcriptional analysis of Arc after whole-cell patch-clamp recordings indicated that increased gene expression correlated with enhancement of synaptic transmission by BDNF. Increased expression in vitro predicted elevations in vivo: VGF and the IEGs increased after trace eyeblink conditioning, a hippocampal-dependent learning paradigm. VGF protein was also upregulated by BDNF treatment and was expressed in a punctate manner in dissociated hippocampal neurons. Collectively, these findings suggested that the VGF neuropeptides may regulate synaptic function. We found a novel function for VGF by applying VGF peptides to neurons. C-terminal VGF peptides acutely increased synaptic charge in a dose-dependent manner, whereas N-terminal peptide had no effect. These observations indicate that gene profiling in vitro can reveal new mechanisms of synaptic strengthening associated with learning and memory.

Isolation of a novel rat neural progenitor clone that expresses Dlx family transcription factors and gives rise to functional GABAergic neurons in culture.

Gamma-aminobutyric acid (GABA) ergic interneurons are lost in conditions including epilepsy and central nervous system injury, but there are few culture models available to study their function. Toward the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP(+) rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2, they exhibit varying patterns of differentiation. Transcriptional profiling and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5, and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In cocultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express ßIII-tubulin, and staining for synaptophysin and vesicular GABA transporter were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses.

Cytoplasmic ATM in neurons modulates synaptic function.

ATM is a PI 3-kinase involved in DNA double-strand break repair. ATM deficiency leads to ataxia-telangiectasia (A-T), a syndrome of cancer susceptibility, hypersensitivity to ionizing radiation, immune deficiency, and sterility [1, 2]-phenotypes that can straightforwardly be attributed to a defective response to DNA damage. Yet patients with A-T also suffer from ataxia, speech defects, and abnormal body movements [3-5]-neurological phenotypes whose origins remain largely unexplained. Compounding the discordance, Atm mutations in mouse interfere with DNA repair but have only mild neurological symptoms [6-9], suggesting that the link between DNA damage and the death of neurons can be broken [10-12]. We find that in neurons, ATM protein has a substantial cytoplasmic distribution. We show that in Atm(tm1Awb) mice, hippocampal long-term potentiation is significantly reduced, as is the rate of spontaneous vesicular dye release, suggesting a functional importance of cytoplasmic ATM. In the cytoplasm, ATM forms a complex with two synaptic vesicle proteins, VAMP2 and synapsin-I, both of which must be phosphorylated to bind ATM. Also, cytoplasmic ATM physically associates with the homologous PI 3-kinase, ATR. The neurological symptoms of ataxia-telangiectasia may thus result from defective nonnuclear functions of ATM not associated with DNA repair.

Endocytosis and membrane potential are required for HeLa cell uptake of R.I.-CKTat9, a retro-inverso Tat cell penetrating peptide.

Cell-penetrating peptides (CPPs) can enter many types of cells and have become useful tools for introducing a variety of cargo such as exogenous peptides, proteins, and nucleic acids into cultured cells in vitro. Tat CPPs derived from the HIV-1 Tat protein are the most widely used among the arginine-rich CPPs. Even though CPPs hold considerable promise for drug delivery, poor biological stability and high in vivo clearance may limit their effectiveness for delivering cargo. Therefore, we utilize a retro-inverso form of a Tat peptide, R.I.-CKTat9, which is proteolytically stable. In the current study, the cellular entry mechanism of this arginine-rich CPP is investigated. Fluorescently labeled R.I.-CKTat9 entered HeLa cells in a concentration- and energy-dependent manner demonstrating both diffuse and punctate (vesicular) appearance inside the cells. The labeled R.I.-CKTat9 colocalized with labeled transferrin in the punctate structure, suggesting that the peptide enters HeLa cells by clathrin-dependent endocytosis. Incubation of cells with an isotonic/high K(+) buffer (KPBS) or an NH(4)Cl solution abolished the diffuse but not the punctate fluorescence, suggesting that membrane potential plays a critical role. This result also suggests that the flux originates from the endosome, not the extracellular space, and relies on the acidity of the endosome. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain function and endocytosis inhibitors greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a single route of endocytosis and subsequent endosomal escape. Since cells in the mitotic (M) phase shut down endocytosis but maintain plasma membrane potential, this property was used to further confirm the endocytic mechanism. Direct measurement of plasma membrane potential confirmed its persistence in M phase arrested HeLa cells. Consistent with our working hypothesis, these cells did not show any vesicular nor diffuse fluorescence of labeled R.I.-CKTat9, providing compelling evidence for the sequential steps of endocytosis and endosomal escape. Binding of labeled R.I.-CKTat9 to the surface of HeLa cells at 0 degrees C was reduced under the mildly acidic conditions of early endosomes, suggesting an acidity-dependent endosomal escape mechanism. Overall, these results indicate that both endocytosis and membrane potential are required for R.I.-CKTat9 entry into HeLa cells and suggest that translocation occurs at the endosomal membrane.

BDNF modulation of NMDA receptors is activity dependent.

Brain-derived neurotrophic factor (BDNF), a potent modulator of synaptic transmission, is known to influence associative synaptic plasticity and refinement of neural connectivity. We now show that BDNF modulation of glutamate currents in hippocampal neurons exhibits the additional property of use dependence, a postsynaptic mechanism resulting in selective modulation of active channels. We demonstrate selectivity by varying the repetition rate of iontophoretically applied glutamate pulses during BDNF exposure. During relatively high-frequency glutamate pulses (0.1 Hz), BDNF application elicited a doubling of the glutamate current. During low-frequency pulses (0.0033 Hz), however, BDNF evoked a dramatically diminished response. This effect was apparently mediated by calcium because manipulations that prevented elevation of intracellular calcium largely eliminated the action of BDNF on glutamate currents. To confirm N-methyl-D-aspartate (NMDA) receptor involvement and assess spatial requirements, we made cell-attached single-channel recordings from somatic NMDA receptors. Inclusion of calcium in the pipette was sufficient to produce enhancement of channel activity by BDNF. Substitution of EGTA for calcium prevented BDNF effects. We conclude that BDNF modulation of postsynaptic NMDA receptors requires concurrent neuronal activity potentially conferring synaptic specificity on the neurotrophin's actions.

Functional differentiation of a clone resembling embryonic cortical interneuron progenitors.

We have generated clones (L2.3 and RG3.6) of neural progenitors with radial glial properties from rat E14.5 cortex that differentiate into astrocytes, neurons, and oligodendrocytes. Here, we describe a different clone (L2.2) that gives rise exclusively to neurons, but not to glia. Neuronal differentiation of L2.2 cells was inhibited by bone morphogenic protein 2 (BMP2) and enhanced by Sonic Hedgehog (SHH) similar to cortical interneuron progenitors. Compared with L2.3, differentiating L2.2 cells expressed significantly higher levels of mRNAs for glutamate decarboxylases (GADs), DLX transcription factors, calretinin, calbindin, neuropeptide Y (NPY), and somatostatin. Increased levels of DLX-2, GADs, and calretinin proteins were confirmed upon differentiation. L2.2 cells differentiated into neurons that fired action potentials in vitro, and their electrophysiological differentiation was accelerated and more complete when cocultured with developing astroglial cells but not with conditioned medium from these cells. The combined results suggest that clone L2.2 resembles GABAergic interneuron progenitors in the developing forebrain.

Developmental and activity-dependent regulation of ARMS/Kidins220 in cultured rat hippocampal neurons.

Neurotrophin activation of Trk receptors elicits diverse effects on neuronal survival, differentiation, and synaptic plasticity. One of the central questions is how specificity is encoded in neurotrophin receptor signaling and actions. A unique downstream protein is the Ankyrin-Repeat Rich Membrane Spanning (ARMS)/Kinase D-interacting substrate-220 kDa (Kidins220), a very abundant scaffold protein in the hippocampus. To determine the roles of ARMS/Kidins220 in hippocampal neurons, we have analyzed the effects of synaptic activity upon the regulation and distribution of ARMS/Kidins220. At early times in vitro (<7 DIV), synaptic activity was low and ARMS/Kidins220 levels were high. As synaptic activity and markers for synapse maturation, such as PSD-95, increased, ARMS/Kidins220 significantly decreased to a plateau by later times in vitro (>12 DIV). Immunocytochemistry showed ARMS/Kidins220 to be concentrated at the tips of growing processes in immature cultures, and more diffusely distributed in older cultures. To examine the apparent inverse relationship between activity and ARMS/Kidins220 levels, neuronal firing was manipulated pharmacologically. Chronic exposure to TTX increased ARMS/Kidins220 levels, whereas bicuculline caused the opposite effect. Moreover, using shRNA to decrease ARMS/Kidins220 levels produced a corresponding increase in synaptic activity. We find that ARMS/Kidins220 may function in neuronal development as an indicator and potentially as a homeostatic regulator of overall synaptic strength in hippocampal neurons.

The neuronal beta 4 subunit increases the unitary conductance of L-type voltage-gated calcium channels in PC12 cells.

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.