Calmodulin and Ca2+ in normal and transformed cells

Numerous lines of evidence implicate calcium and calmodulin (CaM) as regulators of cell growth and functional differentiation. In light of this evidence, several studies of the possible involvement of the CaM system in cellular transformation by RNA and DNA tumor viruses have been carried out. This paper summarizes the evidence linking calcium and CaM to the regulation of cell growth and critically examines the evidence that increases in CaM levels occur in transformed versus normal cells. A nontraumatic method for synchronizing both normal and transformed chick fibroblasts is presented. This method is utilized in a comparison of CaM level throughout the cell cycle of Rous sarcoma virus transformed and normal chick embryo fibroblasts. These studies best support the hypothesis that the observed differences in CaM levels between transformed and normal cultures under optimal growth conditions may largely reflect differences in the proportion of cells in a dividing versus a nondividing state.     

Characterization of Ca2+-stimulated secretion in permeable GH3 pituitary cells

In this report, the secretory response to Ca2+ in GH3 rat pituitary cells permeabilized by electric field discharge has been compared in both magnitude and Ca2+ sensitivity to prolactin (PRL) release from intact GH3 cells. The half-maximally effective [Ca2+] for stimulating PRL release in permeable cells was approximately 0.5 microM, and maximal stimulation was obtained at 3-10 microM Ca2+. The magnitude of Ca2+ stimulation in permeable cells was in the same range as that obtained from an equal number of intact cells stimulated by depolarizing K+. Moreover, the Ca2+ sensitivity of PRL release in intact GH3 cells (measured by Quin 2 fluorescence) closely resembled the Ca2+ sensitivity determined in permeable cells. Release of a sulfated proteoglycan whose release is stimulated by secretagogues in intact cells was stimulated by Ca2+ in permeable cells with the same Ca2+ sensitivity as for PRL release. Maximal Ca2+ stimulation of PRL release in permeable cells required the addition of MgATP. Other energy sources (ADP, GTP, and inorganic phosphate) also supported Ca2+-stimulated secretion but were less effective. The above results indicated that PRL release from permeable cells resembles the physiological process in intact cells. The permeable cell system should prove useful in investigating the mechanism mediating the effect of Ca2+ on secretion, although our studies with pharmacological agents have so far proved inconclusive. Among calmodulin antagonists tested, only trifluoroperazine inhibited Ca2+-stimulated secretion, whereas pimozide and calmidazolium did not.     

Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells

To determine whether hormone synthesis by the GH4C1 pituitary cell line could be regulated by specifically modulating the movement of Ca2+ through voltage-sensitive channels, we have compared the effects of the dihydropyridine Ca2+ channel agonist BAY K8644 and the antagonist nimodipine on hormone production and Ca2+ current in these cells. BAY K8644 elicited, after a 10-15-h lag, a dose-dependent increase in prolactin (PRL) production as determined by measurements of total intracellular and secreted hormone. Over a 72-h period, GH4C1 cells incubated with 300 nM BAY K8644 produced 2-3 times as much total PRL as control cells. The effect on PRL was specific, since BAY K8644 did not increase growth hormone production, cell growth rate, or total cell protein. Exposing GH4C1 cells to BAY K8644 for short periods, up to 90 min, did not induce the delayed increase in PRL production observed with longer incubations. The effects of nimodipine were opposite to those of the Ca2+ channel agonist. PRL production was reduced 85% during 48-h treatment with 200 nM nimodipine, whereas growth hormone production was decreased less than 15%, and cell growth and total protein were unaffected. The actions of these two drugs on PRL production were well correlated with their effects on GH4C1 Ca2+ currents as measured by whole-cell patch-clamp recordings. BAY K8644 enhanced the magnitude of the peak Ca2+ current and shifted the current-voltage relationship such that Ca2+ channels were activated at less depolarized potentials. Nimodipine potently inhibited Ca2+ movement through the non-inactivating channel, while it antagonized the increases elicited by BAY K8644. These results indicate that PRL synthesis by GH4C1 cells can be specifically regulated by agents that enhance or block the movement of Ca2+ through voltage-sensitive channels. They also suggest that hormone synthesis by a secretory cell may be coupled to electrical activity by the opening of Ca2+ channels.     

Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration: studies on Ca2+ channels in rat parotid cells

1. Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration [( Ca2+]i)were studied using quin2. 2. Nicardipine (NIC), diltiazem (DIL) and verapamil (VER) had no effect on the rise in [Ca2+]i evoked by carbachol. Methoxamine-elevated [Ca2+]i was inhibited by VER but not by NIC and DIL. 3. All Ca2+ antagonists tested produced a decline of [Ca2+]i elevated by isoproterenol to the resting level. 4. The addition of 30 mM K+ gradually elevated [Ca2+]i in normal and Ca2+-free media, but it did not increase 45Ca2+ uptake into cells. BAY K 8644 did not increase [Ca2+]i. 5. We suggest that voltage-sensitive Ca2+ channels are lacking and that at least 2 distinct receptor-operated Ca2+ channels exist in rat parotid cells.     

Phenolic antioxidants: potent inhibitors of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

Bis(2-hydroxy-3-tert-butyl-5-methylphenyl)methane (bis-phenol) is the most potent inhibitor of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum yet identified. The compound behaves as a reversible, tight-binding inhibitor with apparent Ki = 0.3 microM. Butylated hydroxytoluene, butylated hydroxyanisole, and 4-nonylphenol are also effective inhibitors. These observations are of particular interest in light of the widespread use of such phenolic antioxidants and stabilizers in the food industry and in the manufacture of rubbers and plastics and the ease with which the compounds are extracted into organic solvents.     

Regulation of hormone secretion and cytosolic Ca2+ by extracellular Ca2+ in parathyroid cells and C-cells: role of voltage-sensitive Ca2+ channels

The two dihydropyridine enantiomers, (+)202-791 and (-)202-791, that act as voltage-sensitive Ca2+ channel agonist and antagonist, respectively, were examined for effects on cytosolic Ca2+ concentrations ([Ca2+]i) and on hormones secretion in dispersed bovine parathyroid cells and a rat medullary thyroid carcinoma (rMTC) cell line. In both cell types, small increases in the concentration of extracellular Ca2+ evoked transient followed by sustained increases in [Ca2+]i, as measured with fura-2. Increases in [Ca2+]i obtained by raised extracellular Ca2+ were associated with a stimulation of secretion of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in rMTC cells, but an inhibition of secretion of parathyroid hormone (PTH) in parathyroid cells. The Ca2+ channel agonist (+)202-791 stimulated whereas the antagonist (-)202-791 inhibited both transient and sustained increases in [Ca2+]i induced by extracellular Ca2+ in rMTC cells. Secretion of CT and CGRP was correspondingly enhanced and depressed by (+)202-791 and (-)202-791, respectively. In contrast, neither the agonist nor the antagonist affected [Ca2+]i and PTH secretion in parathyroid cells. Depolarizing concentrations of extracellular K+ increased [Ca2+]i and hormone secretion in rMTC cells and both these responses were potentiated or inhibited by the Ca2+ channel agonist or antagonist, respectively. The results suggest a major role of voltage-sensitive Ca2+ influx in the regulation of cytosolic Ca2+ and hormones secretion in rMTC cells. Parathyroid cells, on the other hand, appear to lack voltage-sensitive Ca2+ influx pathways and regulate PTH secretion by some alternative mechanism.     

Characterization of phorbol ester- and diacylglycerol-stimulated secretion in permeable GH3 pituitary cells. Interaction with Ca2+

Prolactin (PRL) release in permeable GH3 pituitary cells was stimulated by the protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). Both agents stimulated secretion at 10 nM Ca2+, but higher [Ca2+] (greater than 0.1 microM) potentiated TPA and OAG action. Maximal potentiation occurred at 1 microM calculated free Ca2+, and a similar value was obtained when the cytoplasmic [Ca2+] was measured with the Ca2+-sensitive dye Quin 2. Release of a secretory sulfated proteoglycan was also stimulated by TPA and OAG in permeable GH3 cells, with characteristics similar to those for PRL release. Trifluoroperazine, polymyxin B, neomycin, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate all inhibited both TPA- and Ca2+-stimulated PRL release, but in each case the half-maximal inhibitory concentrations were approximately 2-fold higher for TPA-stimulated release compared to Ca2+-stimulated release. Thyrotropin-releasing hormone (TRH) and guanosine 5'-Q-thiotriphosphate, which stimulate polyphosphoinositide breakdown in permeable cells, were found to be only weak stimulators of PRL release, compared to TPA and exogenous diacylglycerol. However, a much stronger effect of TRH was seen if cells were briefly treated with TRH prior to permeabilization. PRL release from TRH-pretreated permeable cells resembled TPA- and OAG-stimulated secretion, with [Ca2+] greater than 0.1 microM potentiating the effect of TRH pretreatment. These studies support the hypothesis that PRL release in GH3 cells can be stimulated directly by a diacylglycerol-activated secretory mechanism whose activity is modulated by [Ca2+].     

Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells

Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.     

Na and Ca channels in a transformed line of anterior pituitary cells

The ionic conductances of GH3 cells, a transformed line from rat anterior pituitary, have been studied using the whole-cell variant of the patch-clamp technique (Hamill et al., 1981). Pipettes of very low resistance were used, which improved time resolution and made it possible to control the ion content of the cell interior, which equilibrated very rapidly with the pipette contents. Time resolution was further improved by using series resistance compensation and "ballistic charging" of the cell capacitance. We have identified and partially characterized at least three conductances, one carrying only outward current, and the other two normally inward. The outward current is absent when the pipette is filled with Cs+ instead of K+, and has the characteristics of a voltage-dependent potassium conductance. One of the two inward conductances (studied with Cs+ inside) has fast activation, inactivation and deactivation kinetics, is blocked by tetrodotoxin (TTX), and has a reversal potential at the sodium equilibrium potential. The other inward current activates more slowly and deactivates with a quick phase and a very slow phase after a short pulse. Either Ca++ or Ba++ serves as current carrier. During a prolonged pulse, current inactivates fairly completely if there is at least 5 mM Ca++ outside, and the amplitude of the current tails following the pulse diminishes with the time course of inactivation. When Ba++ entirely replaces Ca++ in the external medium, there is no inactivation, but deactivation kinetics of Ca channels vary as pulse duration increases: the slow phase disappears, the fast phase grows in amplitude. Inactivation (Ca++ outside) is unaltered by 50 mM EGTA in the pipette: inactivation cannot be the result of internal accumulation of Ca++.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

Prolactin secretion in permeable GH3 pituitary cells is stimulated by Ca2+ and protein kinase C activators

We have used GH3 cells permeabilized by electric field discharge to examine the effects of Ca2+ and protein kinase C activators (phorbol ester and diacylglycerol) on prolactin (PRL) release. Ca2+ was found to stimulate PRL release approximately 4 fold at 3 microM Ca2+ with a half-maximal response at approximately .5 microM estimated free Ca2+. 12-O-tetradecanoyl phorbol-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol stimulated PRL release throughout a range of Ca2+ concentrations (1 nM -3 microM), but stimulation was greater at higher Ca2+ concentrations (.1 microM to 1 microM). Both agents decreased by 1.8 fold the apparent [Ca2+] at which half-maximal stimulation of secretion occurred. Quin 2 was used to measure the free [Ca2+] of intact and permeable cells; PRL secretion at a free [Ca2+] corresponding to resting cytoplasmic [Ca2+] was 10% of maximal, while secretion at the [Ca2+] corresponding to the Ca2+ spike induced by thyrotropin-releasing hormone was approximately 25% of maximal.     

Differential effects of dopamine antagonists on prolactin secretion from cultured rat pituitary cells.

Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing $\text{in vitro}$ H³-haloperidol binding in rat striatum reported by others (10). Concentration-effect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above 25nM. When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an $\text{in vitro}$ test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.     

Deprivation of Na+, Ca2+ and Mg2+ from the extracellular solution increases cytosolic Ca2+ and stimulates catecholamine secretion from cultured bovine adrenal chromaffin cells

We report here that exposing cultured chromaffin cells to a low ionic strength medium (with sucrose in place of NaCl to maintain osmolarity) can induce a marked elevation in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine (CA) release. To determine the underlying mechanism, we first studied the effects of low [Na+]o on single cell [Ca2+]i (using fluo-3 as Ca2+ indicator) and CA release from many cells. In a Mg2+ and Ca2+-deficient medium, lowering the external concentration of Na2+ ([Na+]o) evoked CA secretion preceded by a transitory [Ca2+]i rise, the amplitude of which was inversely related to [Na+]o. By contrast, in the presence of either [Ca2+]o (2 mM) and [Mg2+]o (1.4 mM) or [Mg2+]o alone (3.4 mM), lowering the ionic strength was without effect. Furthermore, in a physiologic [Na+]o, [Ca2+]o and [Mg2+]o medium, two or three consecutive applications of the cholinergic agonist oxotremorine-M (oxo-M) consistently evoked a substantial [Ca2+]i rise. By contrast, consecutive applications of oxo-M in a Ca2+-deficient medium failed to evoke a rise in [Ca2+]i after the first exposure to the agonist. To clarify the underlying mechanism, we measured and compared the effects of low [Na+]o and the cholinergic agonists nicotine and oxo-M on changes in [Ca2+]i; we studied the effects of these agonists on both membrane potential, Vm (under current clamp conditions), and [Ca2+]i by single cell microfluorimetry (indo-1 as Ca2+ indicator). We observed that, in the presence of [Ca2+]o and [Mg2+]o, lowering [Na+]o had no effect on Vm. In a Ca2+-deficient medium, lowering [Na+]o depolarized the membrane from ca. –60 to –10 mV. As expected, we found that nicotine (10 M) depolarized the membrane (from ca. –60 to –20 mV) and simultaneously evoked a substantial [Ca2+]i rise that was [Ca2+]o-dependent. However, contrary to our expectations, we found that the muscarinic agonist oxo-M (50 M) also depolarized the membrane and induced an elevation in [Ca2+]i. Furthermore, both signals were blocked by D-tubocurarine, insinuating the nicotinic character of oxo-M in adrenal chromaffin cells from bovine. These results suggest that both nicotine and oxo-M stimulate Ca2+ entry, probably through voltage-gated Ca2+-channels. We also show here that oxo-M (and not low [Na+]o) stimulates phosphoinositide turnover.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

Endothelin-induced, long lasting, and Ca2+ influx-independent blockade of intrinsic secretion in pituitary cells by Gz subunits

The G protein-coupled receptors in excitable cells have prominent roles in controlling Ca2+-triggered secretion by modulating voltage-gated Ca2+ influx. In pituitary lactotrophs, spontaneous voltage-gated Ca2+ influx is sufficient to maintain prolactin release high. Here we show that endothelin in picomolar concentrations can interrupt such release for several hours downstream of spontaneous and high K+-stimulated voltage-gated Ca2+ influx. This action occurred through the Gz signaling pathway; the adenylyl cyclase-signaling cascade could mediate sustained inhibition of secretion, whereas rapid inhibition also occurred at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. In a nanomolar concentration range, endothelin also inhibited voltage-gated Ca2+ influx through the G i/o signaling pathway. Thus, the coupling of seven-transmembrane domain endothelin receptors to Gz proteins provided a pathway that effectively blocked hormone secretion distal to Ca2+ entry, whereas the cross-coupling to G i/o proteins reinforced such inhibition by simultaneously reducing the pacemaking activity.     

Simultaneous monitoring of Zn2+ secretion and intracellular Ca2+ from islets and islet cells by fluorescence microscopy

A method for simultaneously imaging Zn2+ secretion and intracellular Ca2+ at beta-cell clusters and single islets of Langerhans was developed. Cells were loaded with the Ca2+ indicator Fura Red, incubated in buffer containing the Zn2+ indicator FluoZin-3, and imaged via laser scanning fluorescence confocal microscopy. FluoZin-3 and Fura Red are excited at 488 nm and emit at 515 and 665 nm, respectively. Zn2+, which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product. Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca2+ and Zn2+ secretion that were correlated with each other and were synchronized among cells. In single islets, spatially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed. Fura Red did not yield detectable Ca2+ signals at islets. For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially illuminating the islets with 340, 380, and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca2+ and secretion to be observed with approximately 2 s temporal resolution. This method should be useful for studying Ca2+ secretion coupling and any application, requiring rapid assays of secretion.     

Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca²⁺ release channel (in the membrane of internal Ca²⁺ stores) and the dihydropyridine receptor (DHPR)-Ca²⁺ channel (in the plasma membrane). To ensure expression of functional L-type Ca²⁺ channels, we expressed α₂, β, and γ DHPR subunits and a chimeric DHPR α₁ subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca²⁺ transient (CaT) in the absence of extracellular Ca²⁺ ([Ca²⁺]_o). However, in the presence of [Ca²⁺]_o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca²⁺]_i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca²⁺ influx through Ca²⁺ channels, whereas the second component resulted from Ca²⁺ release through the RyR expressed in the membrane of internal Ca²⁺ stores. However, the onset and the rate of Ca²⁺ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca²⁺ current. These results suggest that the skeletal muscle RyR isoform supports Ca²⁺-induced Ca²⁺ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.