Extrinsic regulation of T-type Ca(2+) channel expression in chick nodose ganglion neurons

Functional expression of T-type Ca(2+) channels is developmentally regulated in chick nodose neurons. In this study we have tested the hypothesis that extrinsic factors regulate the expression of T-type Ca(2+) channels in vitro. Voltage-gated Ca(2+) currents were measured using whole-cell patch clamp recordings in E7 nodose neurons cultured under various conditions. Culture of E7 nodose neurons for 48 h with a heart extract induced the expression of T-type Ca(2+) channels without any significant effect on HVA currents. T-type Ca(2+) channel expression was not stimulated by survival promoting factors such as BDNF. The stimulatory effect of heart extract was mediated by a heat-labile, trypsin-sensitive factor. Various hematopoietic cytokines including CNTF and LIF mimic the stimulatory effect of heart extract on T-type Ca(2+) channel expression. The stimulatory effect of heart extract and CNTF requires at least 12 h continuous exposure to reach maximal expression and is not altered by culture of nodose neurons with the protein synthesis inhibitor anisomycin, suggesting that T-type Ca(2+) channel expression is regulated by a posttranslational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of heart extract and CNTF suggesting that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Heart extract- or CNTF-evoked stimulation of T-type Ca(2+) channel expression is blocked by the Jak/STAT and MAP kinase blockers, AG490 and U0126, respectively. This study provides new insights into the electrical differentiation of placode-derived sensory neurons and the role of extrinsic factors in regulating the functional expression of Ca(2+) channels.     

T-type Ca(2+) channels mediate neurotransmitter release in retinal bipolar cells

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.     

Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H

Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents.     

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

Cloning and functional expression of novel N-type Ca(2+) channel variants

Voltage-dependent Ca(2+) channels are structurally and functionally diverse. As Ca(2+) currents recorded from embryonic chick dorsal root ganglion (DRG) neurons differ significantly from their mammalian counterparts, information on the primary sequence of the chick channels will help define the structural underpinnings of Ca(2+) channel function. Here, we report the cloning and functional expression of full-length Ca(2+) channel alpha(1B) subunit cDNAs derived from chick DRGs. Two variable regions (A and B) have been identified in the cytoplasmic linker between repeats I and II; a third (C) in the carboxyl terminus extends the open reading frame by 525 nucleotides. The A and C inserts are absent, and the B insert is present in all other class B clones reported to date. The unique shorter channels appear to predominate in DRG neurons. Results represent a requisite first step in defining the structural elements that underlie variations in function and modulation of Ca(2+) channels.     

Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels

T-type Ca(2+) currents have been proposed to be involved in the genesis of spike-and-wave discharges, a sign of absence seizures, but direct evidence in vivo to support this hypothesis has been lacking. To address this question, we generated a null mutation of the alpha(1G) subunit of T-type Ca(2+) channels. The thalamocortical relay neurons of the alpha(1G)-deficient mice lacked the burst mode firing of action potentials, whereas they showed the normal pattern of tonic mode firing. The alpha(1G)-deficient thalamus was specifically resistant to the generation of spike-and-wave discharges in response to GABA(B) receptor activation. Thus, the modulation of the intrinsic firing pattern mediated by alpha(1G) T-type Ca(2+) channels plays a critical role in the genesis of absence seizures in the thalamocortical pathway.     

T-type Ca2+ channel expression in human esophageal carcinomas: a functional role in proliferation

In the present study the role of T-type Ca(2+) channels in cancer cell proliferation was examined. Seventeen human esophageal cancer cell lines were screened for T-type channels using RT-PCR and voltage-clamp recordings. mRNAs for all three T-type channel alpha(1)-subunits (alpha(1G), alpha(1H), and alpha(1I)) were detected in all 17 cell lines: either alpha(1H) alone, alpha(1H) and alpha(1G), or all three T-type alpha(1)-subunits. Eleven cell lines were further subjected to voltage-clamp recordings: one, i.e. the TE8 cell line, was found to exhibit a typical T-type current while others exhibited a minimal or no T-type current. Cell proliferation assays were performed in the presence or absence of T-type channel blocker mibefradil in KYSE150, KYSE180 and TE1 cells expressing mRNA for T-type channel alpha(1)-subunits but lacking T-type current, and TE8 cells exhibiting T-type current. Only TE8 cell proliferation was reduced by mibefradil. Silencing the alpha(1G)-gene that encodes functional T-type Ca(2+) channels in TE8 cells with type-specific shRNA transduction also significantly decreased TE8 cell proliferation. The reduction of cell proliferation in TE8 cells was found to be associated with an up-regulation of p21(CIP1). Moreover, p53 silencing nearly abolished the up-regulation of p21(CIP1) resulting from mibefradil T-type channel blockade. Together, these findings suggest a functional role of T-type channels in certain esophageal carcinomas, and that inhibition of T-type channels reduces cell proliferation via a p53-dependent p21(CIP1) pathway.     

Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release

Electrophysiological studies of neurons reveal different Ca2+ currents designated L-, N-, P-, Q-, R-, and T-type. High-voltage-activated neuronal Ca2+ channels are complexes of a pore-forming alpha 1 subunit of about 190-250 kDa, a transmembrane, disulfide-linked complex of alpha 2 and delta subunits, and an intracellular beta subunit, similar to the alpha 1, alpha 2 delta, and beta subunits previously described for skeletal muscle Ca2+ channels. The primary structures of these subunits have all been determined by homology cDNA cloning using the corresponding subunits of skeletal muscle Ca2+ channels as probes. In most neurons, L-type channels contain alpha 1C or alpha 1D subunits, N-type contain alpha 1B subunits, P- and Q-types contain alternatively spliced forms of alpha 1A subunits, R-type contain alpha 1E subunits, and T-type contain alpha 1G or alpha 1H subunits. Association with different beta subunits also influences Ca2+ channel gating substantially, yielding a remarkable diversity of functionally distinct molecular species of Ca2+ channels in neurons.     

Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.     

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.     

Molecular and functional properties of the human alpha(1G) subunit that forms T-type calcium channels

We describe here several novel properties of the human alpha(1G) subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several alpha(1G) isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: alpha(1G-a) and alpha(1G-b). Northern blot and dot blot analyses indicated that alpha(1G) mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that alpha(1G) mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest alpha(1G) isoform identified to date, alpha(1G-b), resulted in transient, low threshold activated Ca(2+) currents with the expected permeability ratio (I(Sr) > I(Ca) >/= I(Ba)) and channel conductance ( approximately 7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca(2+) channels. This alpha(1G)-related current was inhibited by mibefradil (IC(50) = 2 microM) and weakly blocked by Ni(2+) ions (IC(50) = 148 microM) and amiloride (IC(50) > 1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of -65 to -55 mV. Using neuronal action potential waveforms, we show that alpha(1G) channels produce a massive and sustained Ca(2+) influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca(2+) influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca(2+) channels.     

Two populations of pancreatic islet alpha-cells displaying distinct Ca2+ channel properties

In low or absence of glucose, alpha-cells generate rhythmic action potentials and secrete glucagon. alpha-Cell T-type Ca(2+) channels are believed to be pacemaker channels, which are expected to open near the resting membrane potential (around -60 mV) to initiate a small depolarization. A previous publication, however, showed that alpha-cell T-type Ca(2+) channels have an activation threshold of -40 mV, which does not appear to fulfill their role as pacemakers. In this work, we investigated the Ca(2+) channel characteristics in alpha-cells of mouse-insulin-promoter green-fluorescent-protein (MIP-GFP) mouse. The beta-cells of MIP-GFP were conveniently distinguished as green cells, while immunostaining indicated that the majority of non-green cells were alpha-cells. We found that majority of alpha-cells possessed T-type Ca(2+) channels having an activation threshold of -40 mV; these cells also had high-voltage-activated (HVA) Ca(2+) channels (activation threshold of -20 mV). A novel finding here is that a minority of alpha-cells had T-type Ca(2+) channels with an activation threshold of -60 mV. This minor population of alpha-cells was, surprisingly, devoid of HVA Ca(2+) channels. We suggest that this alpha-cell subpopulation may act as pacemaker cells in low or absence of glucose.     

Specific properties of T-type calcium channels generated by the human alpha 1I subunit

We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.     

A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels

The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

Regulation of neuronal K(+) currents by target-derived factors: opposing actions of two different isoforms of TGFbeta.

The developmental expression of macroscopic Ca(2+)-activated K(+) currents in chick ciliary ganglion neurons is dependent on an avian ortholog of TGFbeta1, known as TGFbeta4, secreted from target tissues in the eye. Here we report that a different isoform, TGFbeta3, is also expressed in a target tissue of ciliary ganglion neurons. Application of TGFbeta3 inhibits the functional expression of whole-cell Ca(2+)-activated K(+) currents evoked by 12 hour treatment with either TGFbeta1 or beta-neuregulin-1 in ciliary ganglion neurons developing in vitro. TGFbeta3 had no effect on voltage-activated Ca(2+) currents. A neutralizing antiserum specific for TGFbeta3 potentiates stimulation of Ca(2+)-activated K(+) currents evoked by a target tissue (iris) extract in cultured ciliary ganglion neurons, indicating that TGFbeta3 is an inhibitory component of these extracts. Intraocular injection of TGFbeta3 causes a modest but significant inhibition of the expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo. Further, intraocular injection of a TGFbeta3-neutralizing antiserum stimulates expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo, indicating that endogenous TGFbeta3 regulates the functional expression of this current. The normal developmental expression of functional Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo is therefore regulated by two different target-derived isoforms of TGFbeta, which produce opposing effects on the electrophysiological differentiation of these neurons.