Kinetics of the TEA and 4-AP sensitive K+ current in the slowly adapting lobster stretch receptor neurone

The kinetics of the TEA and 4-AP sensitive K+ current (IK) in the slowly adapting lobster stretch receptor neurone were investigated in sub- and near-threshold voltage regions using electrophysiological and pharmacological techniques. In dynamic conditions IK was found to display both fast and slow reactions. These were attributed to a Hodgkin-Huxley type of K activation, and a slow type of K inactivation, respectively. The slow K inactivation could be shown to be unrelated to K+ flux dependent changes in intra- and pericellular K+ concentrations. Its stationary voltage dependence was however shifted in a depolarizing direction by increasing, and in hyperpolarizing direction by decreasing the extracellular Ca++ concentration. In view of these findings, and of its kinetic properties, the slow K inactivation was classified as a genuine channel gating process. The process of K activation was too fast for a dynamic analysis with the recording technique available. An estimate of its stationary voltage dependence could however be obtained in a voltage range from about -100 to about -40 mV. The experimental observations were utilized in the formulation of a mathematical model describing the kinetic behaviour of IK in the present preparation based on constant field and state transition theories.     

Modulation of a Ca2+-dependent K+-current by intracellular cAMP in rat thalamocortical relay neurons

Voltage-activated calcium channels in thalamic neurons are considered important elements in the generation of thalamocortical burst firing during periods of electroencephalographic synchronization. A potent counterpart of calcium-mediated depolarization may reside in the activation of calcium-dependent potassium conductances. In the present study, thalamocortical relay cells that were acutely dissociated from the rat ventrobasal thalamic complex (VB) were studied using whole-cell patch-clamp techniques. The calcium-dependent potassium-current (I_K(Ca)) was evident as a slowly activating component of outward current sensitive to the calcium ions (Ca²⁺)-channel blocker methoxyverapamil (10 μM) and to substitution of external calcium by manganese. The I_K(Ca) was blocked by tetraethylammonium chloride (1 mM) and iberiotoxin (100 nM), but not apamin (1 μM). In addition, isolated VB neurons were immunopositive to anti-α_(913–926) antibody, a sequence-directed antibody to the α-subunit of “big” Ca²⁺-dependent K⁺-channel (BK_Ca) channels. Activators of the adenylyl cyclase cyclic adenosine monophosphate (cAMP) system, such as forskolin (20 μM), dibutyryl-cAMP (10 mM) and 3-isobutyl-1-methylxanthine (500 μM), selectively and reversibly suppressed I_K(Ca). These results suggest that a rise in intracellular cAMP level leads to a decrease in a calcium-dependent potassium conductance presumably mediated via BK_Ca type channels, thereby providing an additional mechanism by which neurotransmitter systems are able to control electrogenic activity in thalamocortical neurons and circuits during various states of electroencephalographic synchronization and de-synchronization.     

Voltage clamp on helix pomatia neuronal membrane; Current measurement over a limited area of the soma surface

Summary A method was developed, to measure during a voltage clamp the current which flows through a limited area (10^{4 2}) of the soma membrane. In addition to an intracellular voltage clamp electrode, a fire-polished glass micropipette is placed on the soma surface (with slight suction applied), and only the clamp current through the pipette is measured. This measurement excludes uncontrolled currents from the axon. Alternatively the membrane spot (area under the pipette) can be voltage clamped while the rest of the cell is kept in the resting state. The latter method is an advantage with bigger cells where homogeneous clamp over the whole surface cannot be achieved.     

The mechanism of action of Ba2+ and TEA on single Ca2+-activated K+-channels in arterial and intestinal smooth muscle cell membranes

The interaction of Ba²⁺ and TEA with Ca²⁺-activated K⁺ channels was studied in isolated membrane patches of cells from longitudinal jejunal smooth muscle of rabbit and from guinea-pig small mesenteric artery (100 m external diameter). Ba²⁺ applied from the inside of the membrane did not reduce unit current, except at high concentrations, but channels failed to open for long periods (s). This effect became much stronger when the potential gradient was in a direction driving Ba²⁺ into the channel and was reduced by increasing K⁺ ion concentration on the outside of the membrane. These results are consistent with Ba²⁺ entering the open channel and blocking at a site most of the way through the channel bore. In contrast, TEA and procaine dose-dependently reduced unit current amplitude at all patch potentials and slightly increased mean open time. Their effects were not detectably voltage-dependent and could be explained by TEA and procaine blocking the open channel with a timecourse that was faster than the frequency response of the recording system. The lack of appreciable voltage-dependence suggests that TEA and procaine bind to a site near to the inner mouth of the channel.     

Regulation of K+/Rb+ selectivity and internal TEA blockade by mutations at a single site in K+ pores

A conservative reversion at position 374 in a chimeric K⁺ pore, CHM, switched the preferred ionic conductance from K⁺ to Rb⁺. To understand how selectivity was switched, codons for 18 different amino acids were substituted at position 374 in each of two different K⁺ channels CHM and Kv2.1, the host channel for CHM. After injection of cRNA into Xenopus oocytes, less than half of the substituted mutants expressed functional channels. In both CHM and Kv2.1, channels with the substituted hydrophobic residues Val or Ile expressed Rb⁺-preferring pores while channels with the substituted polar residues Thr or Ser expressed K⁺-preferring pores. Val or Ile stabilized while Thr or Ser destabilized blockade by internal tetraethylammonium (TEA) confirming the importance of hydrophobic interactions for blockade. TEA blockade was dependent upon the charge carrier and was more effective in the presence of the ion having the larger conductance. The results are consistent with a model in which the side chains at position 374 form a filter for K⁺ and Rb⁺ ions and a site for blockade by internal TEA.     

膜环境高温对下丘脑神经元膜上钾通道电压依赖性的影响

目的:观察膜环境高温对下丘脑神经元延迟整流性K^＋通道(I_K)电压依赖性的影响。方法:采用细胞贴附式的膜片钳技术。结果:高温可影响通道的电压依赖性,使电压依赖曲线发生正向移位,过高温度则使电压依赖性减弱甚或丧失。结论:I_K电压依赖性的变化可能参与机体的发热和中暑发病过程。     

Time course of Ca and Ca-dependent K currents during molluscan nerve cell action potentials

The time courses of Ca and Ca-dependent K currents during Ca-dependent action potentials were obtained by recording the membrane currents produced in response to spike-like voltage clamp pulses before and after selective blockade of channels. The Ca current had a biphasic waveform with a first surge and a late, large entry. The Ca-dependent K(Ca) current onset was relatively fast with a peak occurring at half spike repolarization. The fast activation of the K(Ca) current was consecutive to the first Ca entry. It is concluded that K(Ca) currents constitute a powerful spike repolarization mechanism in addition to the voltage-dependent K currents.     

Role of slow delayed rectifier K+-current in QT prolongation in the alloxan-induced diabetic rabbit heart

Aim: In diabetes mellitus, several cardiac electrophysiological parameters are known to be affected. In rodent experimental diabetes models, changes in these parameters were reported, but only limited relevant information is available in other species, having cardiac electrophysiological properties more resembling the human, including the rabbit. The present study was designed to analyse the effects of experimental type 1 diabetes on ventricular repolarization and the underlying transmembrane potassium currents in rabbit hearts. Methods: Diabetes was induced by a single injection of alloxan (145 mg kg⁻¹ i.v.). After the development of diabetes (3 weeks), electrophysiological studies were performed using whole cell voltage clamp and ECG measurements. Results: The QT_c interval in diabetic rabbits was moderately but statistically significantly longer than measured in the control animals (155 ± 1.8 ms vs. 145 ± 2.8 ms, respectively, n = 9–10, P < 0.05). This QT_c-lengthening effect of diabetes was accompanied by a significant reduction in the density of the slow delayed rectifier K⁺ current, I_Ks (from 1.48 ± 0.35 to 0.86 ± 0.17 pA pF⁻¹ at +50 mV, n = 19–21, P < 0.05) without changes in current kinetics. No differences were observed either in the density or in the kinetics of the inward rectifier K⁺ current (I_K1), the rapid delayed rectifier K⁺ current (I_Kr), the transient outward current (I_to) and the L-type calcium current (I_CaL) between the control and alloxan-treated rabbits. Conclusion: It is concluded that type 1 diabetes mellitus, although only moderately, lengthens ventricular repolarization. Diabetes attenuates the repolarization reserve by decreasing the density of I_Ks current, and thereby may enhance the risk of sudden cardiac death.     

Novel transient outward K+ current of mature murine hippocampal neurones

 Hippocampal neurones were freshly isolated from the brain of adult mice and voltage-dependent K⁺ currents were recorded with the whole-cell patch-clamp technique. Three components of transient K⁺ current (IA) were isolated when analyzing data with exponential functions or treating neurones with a variety of voltage protocols and pharmacologic agents. Subtraction of the delayed rectifier current (I _K) from the K⁺ currents elicited after prepulses to –120 mV of varying duration revealed fast (IAf) and slow (IAs) components with decay time constants of 45 ± 8 and 612 ± 140 ms, respectively; the corresponding time constants for the removal of inactivation were 12.3 and 189.6 ms. Both tetraethylammonium and dendrotoxin selectively inhibited IAs. 4-Aminopyridine (4-AP) specifically blocked IAf and 40% of IAs with different affinities. Therefore, the properties of a 4-AP-resistant (IAsR) and a 4-AP-sensitive (IAsS) component of IAs were compared. These data suggest that three distinct subtypes of K⁺ currents contribute to the IA of mature murine hippocampal neurones. Received: 10 April 1996 / Accepted: 11 February 1997     

cAMP-mediated long-term modulation of voltage-dependent K+ channels in cultured colliculi neurons

 Previously, we have described prolonged cAMP-induced inhibition of a K⁺ current in cultured colliculi neurons. The aim of the present study was to characterize the channel responsible for this cAMP-dependent effect. We detected the presence of a non-inactivating voltage-dependent 16-pS K⁺ channel that displayed long-lasting inhibition upon a brief application of cAMP and greater sensitivity to tetraethylammonium than to 4-aminopyridine. In addition to this channel, colliculi neurons express two other voltage-sensitive, non-inactivating K⁺ channels (8 and 49 pS) whose activity is facilitated by a brief application of cAMP, the effect of which is also long-lasting. These results suggest the presence of common sustained cAMP-dependent processes responsible for both up- and down-regulation of these channels in the neurons studied. They indicate that the 16-pS, but not the 8-pS or the 49-pS channels, participates in the cAMP-inhibited macroscopic K⁺ current. Received: 21 April 1998 / Received after revision: 10 July 1998 / Accepted: 5 August 1998     

The effects of temperature and ions on the current—voltage relation and electrical characteristics of a molluscan neurone

1. Current-voltage relations were generated in the Anisodoris giant neurone (G cell) by either current pulses or slow biphasic current ramps.2. Inward-going rectification occurred during hyperpolarization at warm temperatures (10-15 degrees C), but not at cold temperatures (0-5 degrees C) or in the absence of external K.3. Replacing external K with Rb eliminated inward-going rectification in the warm, but produced it in the cold. The removal of external Na, Cl or Ca had no effect upon inward-going rectification.4. At cold temperatures the I-V relation was linear when generated by current pulses, but was non-linear in accordance with the constant field hypothesis when generated by current ramps.5. A high conductance state developed when the membrane was hyperpolarized beyond a critical potential (approximately - 130 mV in the cold, and - 110 mV in the warm) which was dependent upon external Ca, but not upon K, Na or Cl.6. Hysteresis was observed in the ramp-generated I-V relation whenever the cell was polarized into the high conductance state.7. Rectification and the high conductance state appear to involve different mechanisms within the membrane. However, both are dependent upon absolute membrane potential and not the resting potential.8. The axonal-somatic conductance ratio for the G cell was calculated to be between 2 and 10.9. The membrane time constant (200-100 msec) and specific resistance (0.1-1.5 x 10(6) Omega cm(2)) varied with temperature, membrane potential, and external ions in a manner that correlated with changes in the shape of the I-V relation. In addition, the resistance was dependent upon external Ca.10. The K permeability (P(K)), measured during inhibition of inwardgoing rectification, was independent of temperature and membrane potential. However, P(Na) increased with warming.11. The specific capacitance was calculated to be 0.5-1.0 muF/cm(2). The capacitance increased slightly with warming, but was independent of membrane potential and unaffected by reductions in external K or Na.     

Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K⁺, respectively) Ca²⁺-activated K⁺ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca²⁺-activated K⁺ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca²⁺ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca²⁺ is low; at positive membrane potentials, this channel requires Ca²⁺ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca²⁺ in the first minute after excision (activated by 1 M Ca²⁺ at –50 mV). Therefore, it is possible that the Ca²⁺ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca²⁺ concentration and by Ca²⁺ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K _d=17 mM) and is relatively resistant to inhibition by apamin.     

Differential distribution of two Ca2+-dependent and -independent K+ channels throughout receptive and basolateral membranes of bullfrog taste cells

We could identify two types of K⁺ channels, of 80 and 40 pS conductance, respectively, in the bullfrog taste cell membrane using excised and cell-attached configurations of the patch-clamp technique. The taste cell membrane could be divided into four membrane parts — receptive area, apical process, cell body and proximal process. The 80-pS K⁺ channels were dependent on voltage and Ca²⁺ and were located exclusively on the receptive membrane and the apical process membrane. The 40-pS K⁺ channels were independent of voltage and Ca²⁺. The open probability of 40-pS K⁺ channels was decreased by the simultaneous presence of cyclic adenosine monophosphate (cAMP) and adenosine triphosphate (ATP), and the suppressive effect was antagonized by protein kinase inhibitor (PKI). Although 40-pS K⁺ channels were found in a high density on the receptive and apical process membranes, the channels also were present in the other two parts of the taste cell membrane. These results suggest that the two different types of K⁺ channel in the bullfrog taste cells may play different roles in gustatory transduction.     

Ca2+ dependence of small Ca2+-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Single-channel properties of Ca²⁺-activated K⁺ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K⁺ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca²⁺]_i, whereas the mean long open time is independent of [Ca²⁺]_i. At low [Ca²⁺]_i the short-lived open state predominates. At saturating [Ca²⁺]_i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K⁺ current through SK channels is mainly determined by the Ca²⁺-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca²⁺. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca²⁺]_i, the Ca²⁺ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca²⁺ dependence suggest that BK channels are activated by a local high [Ca²⁺]_i associated with Ca²⁺ influx, whereas SK channels may be activated by Ca²⁺ released from internal stores as well.     

Requirement of Na+ and K+ for the action of antidiuretic hormone on water permeability

Previous reports have shown that in the toad bladder the absence of serosal Na+ or K+ inhibits the action of antidiuretic hormone (ADH) on the osmotic permeability (Pf) but not on diffusional permeability (PDW) to water. This dissociation could be due to unstirred layers with low PDW, precluding detection of changes in the PDW of the cells. When serosal Na+ was replaced by choline, Pf (micrometer/s) was inhibited from 201 to 65. In the same conditions, if appropriate corrections were made to allow for the PDW of unstirred layers in the bulk phase and stroma, the PDW (micrometers/s) of the cellular pathway decreased from 19.8 to 15.9. The Pf/PDW ration then became 10 in the presence of serosal Na+ and 11 in its absence. When serosal K+ was deleted Pf decreased from 197 to 127 and PDW (corrected for unstirred layers) from 19.8 to 13.1 The Pf/PDW ratio was 10, both in the presence and absence of serosal K+. In conclusion, it is impossible to estimate the effect of any given experimental manipulation on the ADH-induced increase in PDW and compare it with the effect of Pf unless attention is paid to the restrictions upon measurements of diffusion imposed by unstirred layers. The deletion of Na+ or K+ from the serosa inhibits the effect of ADH on Pf and PDW to the same extent. Therefore, their presence in the serosa is essential for a full effect of ADH on the permeability to water of the apical membrane.     

Inhibitory action of Ca2+ on spontaneous transmitter release at motor nerve terminals in a high K+ solution

The inhibitory effect of a high external Ca²⁺ ([Ca²⁺]_o) on spontaneous transmitter release in a high K⁺ solution (Gage and Quastel 1966; Birks et al. 1968) was studied at the frog neuromuscular junction, based on the hypothesis that an increased intracellular free Ca²⁺ ([Ca²⁺]_i) in the nerve terminal plays a key role in the depression. Three procedures were employed to increase [Ca²⁺]_i; increasing [Ca²⁺]_o, application of caffeine and tetanic nerve stimulation. All of these procedures increased m.e.p.p. frequency in normal Ringer. However, as the basic m.e.p.p. frequency was increased by raising the external K⁺ concentration (7–15 mM), their facilitatory effects on m.e.p.p. frequency decreased, disappeared and eventually reversed to depressant actions. Since a rise in the external K⁺ concentration would increase the steady state level of [Ca²⁺]_i, it is suggested that when the [Ca²⁺]_i is preset at a high level, manipulations so as to further increase [Ca²⁺]_i depress spontaneous release of transmitter. Possible mechanisms for this inhibition was discussed in relation to a question whether or not the rate of spontaneous transmitter release is a monotonic function of [Ca²⁺]_i.     

Differential time course for desensitization to muscarinic effects on K+ and Ca2+ channels

The time course of muscarinic effects on K and Ca currents was investigated at 22–24° C in guinea-pig atrial myocytes, using the whole-cell voltage clamp. At a holding potential of -40 or -50 mV, short exposures to 100 M acetylcholine (ACh) or carbachol (CCh) reproducibly induced outward K currents (I _K,ACh). During long exposures to these agonists, I _K,ACh faded with time. In cells not dialysed with guanosine triphosphate (GTP), I _K,ACh could dissipate completely following 15–20 min of agonist exposure. After agonist washout, lost sensitivity was not recovered. In cells dialysed with GTP (0.2–1 mM), I _K,ACh still faded but normal sensitivity to agonists was restored with washout. Fade of I _K,ACh was not prevented by intracellular heparin or dextran, excluding the involvement of either -adrenergic or muscarinic receptor kinase. I _K,ACh induced by bethanechol or adenosine also faded, and subsequent CCh application after washout revealed a diminished response. Intracellular guanosine-5-o-(3-thiotriphosphate I _K,ACh induced I _K,ACh which faded, and subsequent exposure to CCh was without effect. Equally, after full desensitization with CCh, I _K,ACh failed to induced I _K,ACh. The Ca current (I _Ca) was activated by voltage steps to 0 mV and increased with 1–3 M isoproterenol. This increase could be reversed by CCh, even when I _K,ACh had completely faded. Prolonged muscarinic agonist exposure sometimes also caused fade of the effect on I _Ca, which always occurred after loss of I _K,ACh. The results show that desensitization is heterologous and may involve the guanine nucleotide-binding (G) protein. The differential desensitization to the effects on I _K,ACh and I _Ca suggests the involvement of two different signalling pathways in the muscarinic control of K and Ca channels.     

The action of aldosterone on Na+ and K+ transport in the rat submaxillary main duct

Summary The main excretory duct of the submaxillary gland of normal and adrenalectomized rats was perfused with bicarbonate Ringer's solution and the following values were measured: the transepithelial electrical potential difference, the specific electrical resistance of the epithelium, and the transepithelial net fluxes for Na⁺ and K⁺. From the potential difference and the resistance, the short circuit current was calculated. Following adrenalectomy the short circuit current dropped to about one half, while the electrical resistance increased around twofold and the transepithelial potential difference remained constant. The reduction of short circuit current was accompanied by a 30% reduction of Na⁺ reabsorption whereas K⁺ secretion was only slightly diminished Acute substitution of aldosterone to adrenalectomized animals led to a restitution of the Na⁺ fluxes and showed a tendency to increase K⁺ secretion. Following the administration of Actinomycin D to normal animals, Na⁺ resorption declined as in adrenalectomized rats but K⁺ secretion remained essentially unchanged. From these observations it is concluded that the hypothetical aldosterone-induced proteins act only on Na-resorption and that they may act by both increasing the sodium permeability of the luminal cell membrane and stimulating active Na⁺ transport. The latter effect does not seem to consist of a non specific enhancement of the energy supply since it does not influence the active potassium secretion of the cell.     

Differential effects of apamin on Ca2+-dependent K+ currents in bullfrog sympathetic ganglion cells

In B-type neurones of bullfrog sympathetic ganglia, apamin (10 nM) suppressed the Ca2+-dependent K+ current (IAH) involved in the afterhyperpolarization of an action potential, while it did not affect the Ca2+-dependent K+ current (Ic) underlying the spike repolarization. IAH was further separated into two exponential components which were differentially affected by apamin, voltage and alterations in Ca2+ influx, suggesting the existence of 3 different types of Ca2+-dependent K+ channel in bullfrog sympathetic neurones.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.