Kca3.1 Activation Via P2y2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration.

Disorders in cell signaling mediated by ATP or histamine, activating specific membrane receptors, have been frequently associated with tumorigenesis. Among the elements of response to purinergic (and histaminergic) signaling, ion channel activation controls essential cellular processes in cancer, such as cell proliferation, motility, and death. Here, we studied the effects that ATP had on electrical properties of human ovarian adenocarcinoma cells named SKOV-3. ATP caused increase in intracellular Ca2+ concentration ([Ca2+]i) and, concurrently, it evoked a complex electrical response with a conspicuous outward component. This current was generated through P2Y2 receptor activation and opening of K+ channels, KCa3.1, as indicated by electrophysiological and pharmacological analysis, as well as by immunodetection and specific silencing of P2Y2 or KCa3.1 gene by esiRNA transfection. Low µM ATP concentration increased SKOV-3 cell migration, which was strongly inhibited by KCa3.1 channel blockers and by esiRNA-generated P2Y2 or KCa3.1 downregulation. Finally, in human ovarian tumors, the P2Y2 and KCa3.1 proteins are expressed and co-localized in neoplastic cells. Thus, stimulation of P2Y2 receptors expressed in SKOV-3 cells promotes motility through KCa3.1 activation. Since P2Y2 and KCa3.1 are co-expressed in primary tumors, our findings suggest that they may play a role in cancer progression.

Differential expression of the P2X7 receptor in ovarian surface epithelium during the oestrous cycle in the mouse.

Purinergic signalling has been proposed as an intraovarian regulatory mechanism. Of the receptors responsible for purinergic transmission, the P2X7 receptor is an ATP-gated cationic channel that displays a broad spectrum of cellular functions ranging from apoptosis to cell proliferation and tumourigenesis. In the present study, we investigated the functional expression of P2X7 receptors in ovarian surface epithelium (OSE). P2X7 protein was detected in the OSE layer of the mouse, both in situ and in primary cultures. In cultures, 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) activation of P2X7 receptors increased [Ca(2+)]i and induced apoptosis. The functionality of the P2X7 receptor was investigated in situ by intrabursal injection of BzATP on each day of the oestrous cycle and evaluation of apoptosis 24h using the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end-labelling (TUNEL) assay. Maximum effects of BzATP were observed during pro-oestrus, with the effects being blocked by A438079, a specific P2X7 receptor antagonist. Immunofluorescence staining for P2X7 protein revealed more robust expression during pro-oestrus and in OSE regions behind the antral follicles, strongly supporting the notion that the differences in apoptosis can be explained by increased receptor expression, which is regulated during the oestrous cycle. Finally, P2X7 receptor expression was detected in the OSE layer of human ovaries, with receptor expression maintained in human ovaries diagnosed with cancer, as well as in the human ovarian carcinoma SKOV3 cell line.

Role for ionic fluxes on cell death and apoptotic volume decrease in cultured cerebellar granule neurons.

Recent evidence suggests a major role for ionic fluxes in apoptotic cell death and apoptotic volume decrease. Cerebellar granule neurons (CGN) undergo apoptosis when they are treated with staurosporine or camptothecin (CPT) or when cells are transferred from high extracellular potassium (25 mM KCl [K(+)](e), K25) to low potassium concentration (5 mM KCl [K(+)](e), K5). In this study we described that all three apoptotic conditions induced apoptotic volume decrease in CGN and that two different potassium channel blockers, cesium (Cs(+)) and tetraethylammonium (TEA(+)), prevented the apoptotic volume decrease, caspase-3 activation, nuclear condensation and cell death induced by K5 and CPT, but not by staurosporine. Cs(+) and TEA(+) also blocked membrane currents generated in K5 conditions in CGN. On the other hand, non specific Cl(-) channel blockers such as 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) prevented loss of cell volume induced by K5 or staurosporine. Only the Cl(-) channels blocker but not the K(+) channels blockers protected from staurosporine-induced death of CGN. These data suggest that ionic fluxes play a key role in the activation of the apoptotic volume decrease and apoptotic death of CGN, but the fine mechanism seems to depend on the apoptotic condition.

ARP2 a novel protein involved in apoptosis of LNCaP cells shares a high degree homology with splicing factor Prp8.

The mechanism of apoptosis has been recognized as an important event in processes such as cellular development and homeostasis, as well as degenerative conditions like cancer. Prostate cancer during its advanced stages develops androgen independent cells that ultimately overgrow and promote metastatic events. Our group employing androgen independent LNCaP cells have previously proposed, based on electrophysiological findings, that apoptosis induced cells overexpress a cell death calcium channel-like molecule. Here we report the cloning and expression in Xenopus laevis oocytes of apoptosis regulated protein 2 (ARP2), a protein overexpressed in apoptosis induced LNCaP cells capable to induce calcium inward currents and apoptosis typical morphology changes in oocytes injected with arp2 mRNA. Our results also indicate that clone arp2 cDNA (1.3Kb) shares a 99% homology with a small fragment that corresponds to 18% of the complete sequence of Prp8 cDNA (7.0 Kb), a molecule that codifies for an important protein in the assembly of the spliceosome. We propose that protein ARP2 as a fragment of protein Prp8, corresponds to a molecule with a new function in apoptosis related phenomena.

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells.

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.

Extracts from plants used in Mexican traditional medicine activate Ca(2+)-dependent chloride channels in Xenopus laevis oocytes.

The two-electrode voltage-clamp technique was employed to investigate the effects of chloroform-methanol (1:1) extracts derived from five medicinal plants on Xenopus laevis oocytes. When evaluated at concentrations of 1 to 500 microg/ml, the extracts prepared from the aerial parts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium murale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produced concentration-dependent oscillatory inward currents in the oocytes, while the extract of Gentiana spathacea did not induce any response. The reversal potential of the currents elicited by the active extracts was -17 +/- 2 mV and was similar to the chloride equilibrium potential in oocytes. These ionic responses were independent of extracellular calcium. However, they were eliminated by overnight incubation with BAPTA-AM (10 microM), suggesting that the currents were dependent on intracellular Ca2+ increase. Thus the plant extracts activate the typical oscillatory Ca(2+)-dependent Cl- currents generated in the Xenopus oocyte membrane more probably via a mechanism that involves release of Ca2+ from intracellular reservoirs. These observations suggest that Xenopus oocyte electrophysiological recording constitutes a suitable assay for the study of the mechanisms of action of herbal medicines.

Glycosylated and phosphorylated proteins--expression in yeast and oocytes of Xenopus: prospects and challenges--relevance to expression of thermostable proteins.

Phosphorylation and glycosylation are important posttranslational events in the biosynthesis of proteins. The different degrees of phosphorylation and glycosylation of proteins have been an intriguing phenomenon. Advances in genetic engineering have made it possible to control the degree of glycosylation and phosphorylation of proteins. Structural biology of phosphorylated and glycosylated proteins has been advancing at a much slower pace due to difficulties in using high-resolution NMR studies in solution phase. Major difficulties have arisen from the inherent mobilities of phosphorylated and glycosylated side chains. This paper reviews molecular and structural biology of phosphorylated and glycosylated proteins expressed in eukaryotic expression systems which are especially suited for large-scale production of these proteins. In our laboratory, we have observed that eukaryotic expression systems are particularly suited for the expression of thermostable light-activated proteins, e.g., bacteriorhodopsins and plastocyanins.

Electrophysiological and hemolytic activity elicited by the venom of the jellyfish Cassiopea xamachana.

In this study, we determined hemolysis activity in human and sheep erythrocytes, and characterized the electrical responses in Xenopus oocyte membrane elicited by the venom of the jellyfish Cassiopea xamachana (Cx). The Cx venom produced hemolysis in both species, being more potent on human red cells. The electrophysiological study showed that the Cx venom elicited three different responses in the oocytes. One current was generated in all the oocytes tested and corresponded with a slow inward current (I(Cx)) associated with an increase in membrane conductance. I(Cx) was concentration-dependent and had a reversal potential of -10.3+/-0.4 mV. Ionic substitution studies indicated that the conductive pathway was mainly permeable to cations and non-selective. The oocyte membrane resistance was completely recovered after washout of the venom, this suggested that the effect was due to generation of a specific membrane conductance as opposed to a possible non-specific membrane breakdown. A comparative study with three distinct native cationic channels present in the oocyte membrane [i.e. (1) hemi-gap-junction channels, (2) mechanosensitive channels, and (3) the ouabain-sensitive channel activated by palytoxin], showed that I(Cx) might correspond to opening of mechanosensitive channels or to activation of an unknown cationic channel located in the oocyte membrane. The bioactive fraction eliciting I(Cx) were peptides and was separated from two other peptidic hemolytic fractions by chromatography.

Activation of volume-regulated Cl(-) channels by ACh and ATP in Xenopus follicles.

Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique. In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application. In follicles, the permeability ratios for different anions with respect to Cl- were similar for both ICl,swell and Sin, with a sequence of: SCN- > I- > Br- >= NO3- >= Cl- > gluconate >= cyclamate > acetate > SO42-. Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was approximately 100 % slower at pH 8.0 compared with that at pH 6.0. Lanthanides inhibited ICl, swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 +/- 1.9 microM, while Sin was blocked up to 55 % with an apparent IC50 of 36 +/- 2.6 microM. Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 +/- 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to -20 mV. This single-channel activity was increased by application of ACh or ATP. The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling. All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity, strongly suggest that ICl,swell and Sin currents depend on the opening of the same type or a closely related class of volume-regulated chloride channels.

Muscarinic receptor heterogeneity in follicle-enclosed Xenopus oocytes.

1. Ionic current responses elicited by acetylcholine (ACh) in follicle-enclosed Xenopus oocytes (follicles) were studied using the two-electrode voltage-clamp technique. ACh generated a fast chloride current (Fin) and inhibited K+ currents gated by cAMP (IK,cAMP) following receptor activation by adenosine, follicle-stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs. 2. Inhibition of IK,cAMP was about 100 times more sensitive to ACh than Fin activation; the half-maximal effective concentrations (EC50) were 6.6 +/- 0.4 and 784 +/- 4 nM, respectively. 3. Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 +/- 0.07 nM, whilst the IC50 for IK,cAMP inhibition was 5.9 +/- 0.2 microM. 4. Oxotremorine, a muscarinic agonist, preferentially stimulated IK, cAMP inhibition (EC50 = 15.8 +/- 1.4 microM), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 +/- 0.7 microM. 5. Fin elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell-oocyte coupling. 6. Using reverse transcription-PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in IK,cAMP inhibition. 7. In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype and the other the M3 subtype. These receptors are coupled to distinct membrane mechanisms leading to independent regulation of two membrane conductances.

Cl- currents activated via purinergic receptors in Xenopus follicles.

Ionic currents elicited via purinergic receptors located in the membrane of Xenopus follicles were studied using electrophysiological techniques. Follicles responded to ATP-activating inward currents with a fast time course (F(in)). In Ringer solution, reversal potential (Erev) of F(in) was -22 mV, which did not change with external substitutions of Na- or K+, whereas solutions containing 50 or 5% of normal Cl- concentration shifted Erev to about +4 and +60 mV, respectively, and decreased F(in) amplitude, indicating that F(in) was carried by Cl-.F(in) had an onset delay of approximately 400 ms, measured by application of a brief jet of ATP from a micropipette positioned near the follicle (50 microns). F(in) was inhibited by 50% in follicles pretreated with pertussis toxin. This suggests a G protein-mediated receptor channel pathway. F(in) was mimicked by 2-MeSATP and UTP, the potency order (half-maximal effective concentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP (1,086 nM). All agonists generated Cl- currents and displayed cross-inhibition on the others. F(in) activation by acetylcholine also cross-inhibited F(in)-ATP responses, suggesting that all act on a common channel-activation pathway.

Functional role of follicular cells in the generation of osmolarity-dependent Cl- currents in Xenopus follicles.

1. Osmolarity-dependent (osmo-dependent) ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using the two-microelectrode voltage-clamp technique, combined with intra-oocyte pressure injection of sucrose or polyethylene glycols (PEGs). 2. Intra-oocyte injections of sucrose or PEG (3-25 nmol) generated inward membrane currents (follicles held at -60 mV) associated with an increase in membrane conductance. These currents were carried mainly by chloride ions (ICl(osm)), and were strongly attenuated by increasing the tonicity of the external medium, or by external application of La3+ (0.1-1 mM). 3. The ability to generate ICl(osm) depended on the molecular weight of the injected PEG. Injections of PEG 200 or 300 generated ICl(osm) in 95% of the follicles tested, PEG 600 generated comparable currents in only 20% of the follicles, while similar injections of PEG 1000 did not elicit ICl(osm). 4. Octanol (1-1.5 mM), a gap junction channel blocker, reversibly inhibited 50-90% of the ICl(osm) generated by injections of sucrose or PEG 300. Moreover, sucrose or PEG injections did not elicit ICl(osm) in defolliculated oocytes. 5. It is concluded that an increase in the internal osmolarity of the follicular cells activates a mechanism, probably involving cellular swelling, which leads to the opening of ICl(osm) channels most probably located in the follicular cell membrane.

A monovalent cationic conductance that is blocked by extracellular divalent cations in Xenopus oocytes.

1. Native Xenopus oocytes were voltage clamped and exposed to Ringer solutions containing low concentrations of divalent cations. Oocytes, held at -60 mV, developed a reversible non-inactivating smooth inward current (Ic) associated with an increase in membrane conductance. 2. Ic was selectively carried by cations (Na+, K+), indicating that the current was not the result of a non-specific membrane breakdown, but was due instead to removal of a blocking effect of divalent cations on a specific population of endogenous ionic channels located in the oocyte membrane. 3. The blocking effects of Ca2+ and Mg2+ were voltage dependent, implying action at a binding site within the pore of the cationic channel. For example, the half-maximal inhibition (IC50) of Ic by Ca2+ was 61 microM in oocytes held at -60 mV and 212 microM in oocytes held at 0 mV. 4. The Ic channels could be unblocked by depolarization of the membrane even in the presence of physiological concentrations of Ca2+ or Mg2+. The unblocking of the channels was observed as a slowly developing outward current. 5. The novel cationic current was substantially reduced following in vitro maturation of oocytes by treatment with progesterone (10 microM, 4-5 h). 6. The physiological role of Ic channels remains to be elucidated. Nonetheless, their characteristics explain the ionic basis of the sensitivity of oocytes to reductions in extracellular divalent cations and raise the possibility that the channels play a role in calcium homeostasis.

Osmo-dependent Cl- currents activated by cyclic AMP in follicle-enclosed Xenopus oocytes.

The role of adenosine 3',5'-cyclic monophosphate (cAMP) in generating the osmo-dependent slow inward membrane currents (S(in)) elicited by activation of follicle stimulating hormone (FSH) or acetylcholine (ACh) receptors was studied in voltage-clamped, follicle-enclosed oocytes of Xenopus laevis (follicles). Forskolin (FSK) also generated S(in) currents, and in low concentrations it potentiated the S(in) currents elicited by FSH but not those elicited by ACh. Moreover, intra-oocyte injections of cAMP elicited similar slow inward currents (cAMP-S(in)) that: (i) were carried mainly by chloride ions; (ii) were abolished by defolliculating the oocytes; and (iii) were dependent on the osmolarity of the external medium. Compared with the Ca(2+)-dependent chloride channels that are located in the oocyte membrane; the cAMP-activated S(in) channels were less permeable to I- and Br-, and their current-voltage relation did not rectify strongly at negative potentials. Generation of cAMP-S(in) desensitized the FSH-S(in) currents, but did not have effects on both the S(in) and the fast chloride current (F(in)) specifically elicited by ACh. Furthermore, follicular phospholipase C activation through stimulation of angiotensin II (AII) receptors failed to generate the current responses elicited by ACh. We conclude that cAMP acts as a potent second messenger in generating the osmo-dependent Cl- currents elicited by FSH but not those elicited by ACh. The mechanisms underlying the ACh responses remain unknown. The osmo-dependent chloride channels activated by cAMP may play a role in the control of volume of the follicular cells-oocyte complex.

Novel Cl- currents elicited by follicle stimulating hormone and acetylcholine in follicle-enclosed Xenopus oocytes.

Voltage-clamp techniques were used to study the membrane currents elicited by follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle-enclosed oocytes of Xenopus laevis (follicles). Both agonists caused complex responses that were more evident when the follicles were in hypotonic Ringer solution (HR; 190.4 mosM). In this medium, currents activated by FSH regularly showed three phases whereas currents activated by ACh displayed three to six phases. At a holding potential of -60 mV, FSH, and ACh responses involved combinations of inward and outward currents. Both FSH and ACh responses included a slow smooth inward component that was associated with an increase in membrane conductance, mainly to Cl- (S(in)). This current was strongly dependent on the osmolarity of the external solution: an increase in osmolarity of the HR solution of 18-20 mosM caused a 50% decrease in S(in). In contrast, a fast and transient Cl- current (F(in)) specifically elicited by ACh was not dependent on osmolarity. Both, F(in) and S(in) currents required the presence of follicular cells, since defolliculation using three different methods abolished all the response to FSH and at least four components of the ACh responses. The membrane channels carrying F(in) and oscillatory Cl- currents elicited by stimulation of ACh or serum receptors, were much more permeable to I- and Br- than Cl-, whereas S(in) channels were equally permeable to these anions. Unlike the oscillatory Cl- currents generated in the oocyte itself, S(in) and F(in) currents in follicle-enclosed oocytes were not abolished by chelation of intracellular Ca2+, either with EGTA or BAPTA, which suggests that intracellular Ca2+ does not play a critical role in the activation of these currents. Our experiments show that S(in) and F(in) currents are quite distinct from the previously characterized oscillatory Cl- responses of oocytes. Moreover, the results strongly suggest that the FSH and ACh receptors, the Cl- channels mediating the F(in) and S(in) currents, together with the necessary elements for their activation, are all located in the follicular cells and not in the oocyte. Many aspects of follicular cell physiology in Xenopus laevis, and other species, are regulated by hormones and neurotransmitters, including FSH and ACh. The follicular Cl- currents described in this paper may play an important role in the follicular cell-oocyte development.

mRNA coding for neurotransmitter receptors in a human astrocytoma.

Electrophysiological techniques and Xenopus oocytes were used to study the expression of neurotransmitter receptors encoded by mRNAs isolated from three human glioma cell lines. Oocytes injected with mRNAs from two glioblastoma cell lines did not show electrical responses to the various neurotransmitters tested. In contrast, oocytes injected with mRNA from an astrocytoma cell line (R-111) acquired acetylcholine and glutamate receptors as well as a small number of N-methyl-D-aspartate (NMDA) receptors. Acetylcholine elicited oscillatory Cl- currents that were abolished by muscarinic antagonists. The muscarinic receptors are coupled to the inositol phosphate-Ca2+ receptor-channel coupling system. Glutamate and its analogs kainate, quisqualate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid induced smooth currents. The non-NMDA responses were potently blocked by 6,7-dinitroquinoxaline-2,3 dione. Our results show that human astrocytoma cells contain mRNAs coding for functional acetylcholine and glutamate receptors that have properties similar to those of neurons. In contrast, human glioblastoma cells lacked those mRNAs. These differences might be useful for the development of new diagnostic and therapeutic procedures.

Humoral factors reduce gap junction sensitivity to cytoplasmic pH. II. In vitro manipulations.

Our previous studies demonstrated a diurnal rhythm in the response of gap junctions between crayfish giant axons to acidification and that the response was reduced after eyestalk ablation, sinus gland removal, or visual stress. In this paper we describe experiments to test whether compounds in the circulating hemolymph were responsible for modulation of the responsiveness gap junction channels to intracellular pH. In axons from destalked animals in which the hemolymph had been replaced with normal saline, the maximal junctional resistance after acidification (Rjmax) reached control values. In contrast, Rjmax reached only 30% of control after acidification in axons from animals that had been destalked but not perfused. Hemolymph drawn after eyestalk ablation was tested on axons from control animals. Treatment with hemolymph drawn 1 day after destalking resulted in control Rjmax values, while treatment with hemolymph drawn 7 days after destalking resulted in Rjmax values of only 5-40%. Similarly, pretreatment for 1 h with 100 microM ecdysterone resulted in low Rjmax values. These experimental results suggest that a circulating compound, most likely ecdysterone or a related molecule, regulates the physiological properties of gap junctions from crayfish lateral axons.

Protein phosphorylation and hydrogen ions modulate calcium-induced closure of gap junction channels.

The regulation of the cell-to-cell pathway formed by gap junctions seems to involve the interaction of the junctional channels with either calcium or hydrogen ions, as well as protein phosphorylation and calmodulin. These mechanisms of junctional regulation have been considered to act independently on specific sites of the gap junction protein; however, the possibility that they may be interrelated has not been adequately explored mainly due to the difficulties involved in simultaneous measurement of intracellular cations and protein phosphorylation. To further understanding of mechanisms regulating gap junctions, we have internally perfused coupled lateral axons from crayfish with solutions containing different calcium and hydrogen concentrations under conditions favoring phosphorylation, while monitoring the junctional conductance. We found that calcium ions regulate cell communication probably through a direct interaction with the channel protein. Phosphorylation and low pH do not alter junctional conductance themselves, but appear only to modulate the effects of calcium, possibly by altering the affinity of the channel for calcium. We propose that a combination of free intracellular calcium and protein phosphorylation form an important physiological mechanism regulating intercellular communication.

Calmodulin acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons.

Lateral axons from the abdominal nerve cord of crayfish were internally perfused with the calcium receptor calmodulin (CaM) in solutions with low (pCa greater than 7.0) or high (pCa 5.5) calcium concentrations and studied electrophysiologically and morphologically. Results from these experiments show that when the internal solution contains calcium-activated calmodulin (Ca2+-CaM) the junctional resistance between the axons increases from control values of about 60 to 500-600 k omega in 60 min. In contrast, axons perfused with calmodulin in low calcium solutions maintain their junctional resistance at control levels during the 60-min perfusion. Similar results are obtained when only one or both coupled axons are perfused. The morphological study shows that in the perfused axons the axoplasmic organelles are replaced or grossly perturbed by the perfusion solution up to the region of the synapses. Additionally, in axons perfused with Ca2+-CaM there are regions where the synaptic gap between the membranes decreases from a control 4-6 to 2-3 nm. Both electrophysiological and morphological results can be interpreted as indicating that calcium-activated calmodulin acts directly on the junctional channels to induce their closure.