cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel.

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1).

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.

Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells.

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.

Spontaneous action potentials initiate rhythmic intercellular calcium waves in immortalized hypothalamic (GT1-1) neurons.

GT1-1 cells exhibit spontaneous action potentials and transient increases in intracellular calcium concentration ([Ca2+]i) that occur in individual cells and as spatially propagated intercellular Ca2+ waves. In this study, simultaneous cell-attached patch-clamp recording of action currents (indicative of action potentials) and fluorescence imaging of [Ca2+]i revealed that Ca2+ transients in GT1-1 cells were preceded by a single action current or a burst of action currents. Action currents preceded Ca2+ transients in a similar pattern regardless of whether the Ca2+ transients were limited to the individual cell or occurred as part of an intercellular Ca2+ wave. Both the action currents and Ca2+ transients were abolished by 1 microM tetrodotoxin. Removal of extracellular Ca2+ abolished all spontaneous Ca2+ transients without inhibiting the firing of action currents. Nimodipine, which blocks L-type Ca2+ currents in GT1-1 cells, also abolished all spontaneous Ca2+ signaling. Delivery of small voltage steps to the patch pipette in the cell-attached configuration elicited action currents the latency to firing of which decreased with increasing amplitude of the voltage step. These results indicate that spontaneous intercellular Ca2+ waves are generated by a propagated depolarization, the firing of action potentials in individual cells, and the resulting influx of Ca2+ through L-type Ca2+ channels. These patterns of spontaneous activity may be important in driving the pulsatile release of GnRH from networks of cells.

Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A.

The rabbit cardiac Ca2+ channel (alpha1C) expressed in Xenopus oocytes exhibited a complete run-down of ionic currents when cell-attached patches were excised. The alpha1C channel was expressed alone or was coexpressed with the accessory beta2a or beta1b subunit. The catalytic subunit of protein kinase A (PKAc) and MgATP were capable of delaying the run-down of single-channel currents. In 33% of the alpha1C patches, and 26% of the alpha1C+beta2a patches, inclusion of PKAc in the bath solution delayed the run-down for a maximum of 20 min. In experiments where PKAc in the bath was not sufficient to delay the run-down of channel activity, insertion of the patch back into the oocyte (patch-cramming) could restore channel activity. Gating currents were also measured in the alpha1C+beta1b channel and were not subject to any run-down, even after the complete run-down of ionic currents. The results presented here reveal that PKAc is capable of delaying the run-down of currents in a subset of patches. The patch-cramming results suggest that a cytoplasmic factor, in addition to phosphorylation of the channel (by PKAc), may be involved in the maintenance of channel activity.

Structures and functions of calcium channel beta subunits.

Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.

Unique regulatory properties of the type 2a Ca2+ channel beta subunit caused by palmitoylation.

Beta subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for beta2a, which differs in that it does not support prepulse facilitation of alpha1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal alpha1E Ca2+ channels, and is more effective in blocking inhibition of alpha1E channels by G protein-coupled receptors. We show that the distinguishing properties of beta2a, rather than interaction with a distinct site of alpha1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of beta2a were lost in a mutant that could not be palmitoylated [beta2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three beta2 splice variants differing only in their N-termini.

Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels.

1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

Long lasting facilitation of the rabbit cardiac Ca2+ channel: correlation with the coupling efficiency between charge movement and pore opening.

Facilitation of Ca2+ entry into cells enhances Ca(2+)-activated events such as transmitter release and stimulation of second messenger systems. We have found a long lasting prepulse facilitation (up to 3-fold) of the cardiac Ca2+ channel alpha1Cbeta1b that lasts for tens of seconds without altering current kinetics. The voltage- and time-dependence of the installation of facilitation was characterized as well as the time- and use-dependence of the decay of facilitation. The degree of facilitation was correlated with the coupling efficiency between the charge movement and pore opening channels that were poorly coupled prior to facilitation exhibited the largest facilitation.

Molecular analysis and functional expression of the human type E neuronal Ca2+ channel alpha 1 subunit.

A human brain alpha 1 Ca2+ channel subunit was cloned and expressed in Xenopus laevis oocytes. The open reading frame, encoding 2,312 amino acids, has high homology to the marine ray doe-1, the rat E-type, and the rabbit brain BII alpha 1 subunits. The amino and carboxy termini of this human.E-type alpha 1 subunit (alpha 1E) are most similar to the rabbit BII-1 splice variant, the remainder being colinear with the BII alpha 1 with the exception of two insertions, one of 43 amino acids in the C-terminus and another of 7 amino acids, found also in the rat alpha 1E, between domains II and III. Two potential Ca2+ binding sites are predicted from its primary structure. The expression of inward Ba2+ currents reveals voltage-dependent activation and inactivation measured by the cut-open oocyte vaseline-gap technique, with kinetics that correspond to that of a high-voltage-activated neuronal Ca2+ channel, and pharmacologic properties that resemble those of some low-voltage-activated neuronal Ca2+ currents. The human alpha 1E currents are insensitive to omega-conotoxin-GVIA (1 microM), omega-agatoxin-IVA (200 nM), a synthetic funnel web spider toxin (FTX, 20 microM), and Bay-K8644 (0.5 microM); they are inhibited 20% by high concentrations of methoxyverapamil and diltiazem, 65% by 0.1% crude funnel web spider venom and 100% by Ni2+ (IC50 = 30 nM). Single-channel records show a complex activity pattern with several apparent conductance states, the largest having a conductance of 14 pS.

Effects of a Shaker K+ channel peptide and trypsin on a K+ channel in Necturus enterocytes.

We have previously demonstrated that a synthetic peptide composed of the first 22 amino acids from the NH2-terminus of the Shaker B K+ channel protein deactivates a voltage-dependent K+ channel present in basolateral membrane of Necturus small intestinal epithelial cells reconstituted into planar lipid bilayers (Dubinsky et al. Proc. Natl. Acad. Sci. USA 89: 1770-1774, 1992). We now demonstrate that this peptide interacts with the inner surface of the Necturus channel only when it is in the open or conducting configuration and that this interaction is hindered by tetraethylammonium ion, a well-established blocker of this and other K+ channels. We conclude that this peptide is an open-pore blocker of the Necturus K+ channel as it appears to be in the case of the Shaker B K+ channel. We further demonstrate that trypsin, which abolishes the ability of this peptide to block both the Necturus and the Shaker K+ channels and inhibits spontaneous inactivation of the Shaker K+ channel, also impairs the voltage-gate of the Necturus K+ channel. These findings, and others to be reported in a companion paper, suggest structural homologies between the "inactivation peptide" of the Shaker B K+ channel and the voltage-gate of the Necturus K+ channel.

Reconstitution of a calcium-activated potassium channel in basolateral membranes of rabbit colonocytes into planar lipid bilayers.

A highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989) J. Membrane Biol. 110:147-162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance. Ca2+-activated K+ channel. This channel is highly selective for K+ over Na+ and Cl-, displays voltage-gating similar to "maxi" K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K+ diffusion through the channel involves either the binding of a single K+ ion to a site within the channel or "single-filing" ("multi-ion occupancy"). Channel activity is inhibited by the venom from the scorpion Leiurus quinquestriatus, Ba2+, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed.

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Basolateral membrane vesicles from Necturus enterocytes, highly (greater than 20-fold) enriched in Na+,K+-ATPase, were reconstituted into planar lipid bilayers. The principal channel activity observed is selective for K+ over Na+ and Cl-. This K+ channel is blocked by Ba2+ and Leiurus quinquestriatus venom but is not affected by Ca2+ over the range of 10(-3) to less than 10(-7) M and is not inhibited by charybdotoxin. L. quinquestriatus venom also markedly reduces the conductance of the basolateral membrane of intact villus cells of Necturus small intestine. The open-time probability (Po) of the channel displays a voltage-dependence characteristic of an "inward rectifier"; i.e., the channel inactivates when the basolateral membrane is depolarized and Po increases with increasing hyperpolarization of that barrier. Assuming that similar properties prevail under physiological conditions, this characteristic could provide, in part, an explanation for the parallelism between Na+-pump and K+-leak activities of the basolateral membrane observed in this epithelium. Thus, an increase in rheogenic Na+-pump activity at the basolateral membrane would hyperpolarize that barrier and, in turn, increase the open time of this K+ channel.

Diphenylamine-2-carboxylate blocks Cl(-)-HCO3- exchange in Necturus gallbladder epithelium.

Intracellular microelectrode techniques were employed to study the effects of diphenylamine-2-carboxylate (DPC) on ion transport in Necturus gallbladder epithelium. Under control conditions, addition of DPC to the mucosal bathing solution caused a concentration-dependent, reversible hyperpolarization of both cell membranes with no measurable resistance changes. In addition, DPC caused the following effects, all consistent with inhibition of apical membrane Cl(-)-HCO3- exchange: fall in intracellular Cl- activity (aCli), increase in intracellular pH (pHi), reduction of the changes in aCli and pHi produced by lowering mucosal solution [Cl-], and reduction of the change in pHi produced by lowering mucosal solution [HCO3-]. Similar studies in theophylline-treated preparations indicate that DPC also inhibits anion exchange under these conditions, but has no effect on the apical membrane electrodiffusive Cl- permeability induced by cyclic AMP. Under these conditions, DPC caused cell membrane hyperpolarization but had no effect on the apparent ratio of membrane resistances. In addition, DPC had no effects on the rapid changes in apical membrane voltage elicited by altering mucosal [Cl-], but caused significant reductions of the slower, secondary voltage changes observed in response to changes in mucosal [Cl-], and the changes in aCli and pHi produced by lowering mucosal [Cl-]. Because others have demonstrated that DPC blocks Cl- channels in other epithelia (Distefano, A., M. Wittner, E. Schlatter, H. J. Lang, H. Englert, and R. Greger. Diphenylamine-2-carboxylate, a blocker of the Cl(-)-conductive pathway in Cl(-)-transporting epithelia. Pfluegers++ Arch. 405: S95-S100, 1985), it is possible that the structures of those channels and that induced by cyclic AMP in Necturus gallbladder are different. Because of its relatively high affinity and rapid reversibility, DPC may become useful in studies of anion exchange in other cells.

Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder.

The hypothesis of Cl-/HCO3- exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pHo), intracellular pH (pHi), and Cl- activity (alpha Cli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na+/H+ exchange inhibitor amiloride (10(-3) M) to the superfusate, alkalinization was observed. During prolonged (10 min) exposure to amiloride, no significant pHo change occurred. Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na+ in the superfusate (TMA+ substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of Cl- (cyclamate or sulfate substitution) caused a short-lived increase in the rate of luminal acidification, followed by a return to control values (10-30 min). Upon re-exposure to Cl-, there was a transient reduction of luminal acidification. The initial increase in acidification produced by Cl- removal was partially inhibited by SITS (0.5 mM). The pHi increased rapidly and reversibly when the Cl- concentration of the mucosal bathing solution was reduced to nominally 0 mM. The pHi changes were larger in 10 mM HCO3-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO3- is transported in exchange for Cl-. In both HEPES- and HCO3-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of alpha Cli. These results support the hypothesis of apical membrane Cl-/HCO3- exchange, which can be dissociated from Na+/H+ exchange and operates under control conditions. The coexistence at the apical membrane of Na+/H+ and Cl-/HCO3- antiports suggests that NaCl entry can occur through these transporters.