The alpha subunit of the GTP binding protein activates muscarinic potassium channels of the atrium.

It has been debated whether the potassium channel of the atrium is activated by the alpha subunit or by the beta gamma subunits of guanine nucleotide binding (G) proteins, which dissociate on activation with guanosine triphosphate (GTP). Therefore, the channel-activating effectiveness of these subunits on isolated guinea pig atrial cells was tested. The activated alpha K subunit from human erythrocytes activated the channel in subpicomolar concentrations. The beta gamma dimer from bovine brain activated the channel in nanomolar concentrations. These results support the view that, physiologically, the alpha subunit activates the channel.

Intracellular pH modulates the availability of vascular L-type Ca2+ channels.

L-type Ca2+ channel currents were recorded from myocytes isolated from bovine pial and porcine coronary arteries to study the influence of changes in intracellular pH (pHi). Whole cell ICa fell when pHi was made more acidic by substituting HEPES/NaOH with CO2/bicarbonate buffer (pHo 7.4, 36 degrees C), and increased when pHi was made more alkaline by addition of 20 mM NH4Cl. Peak ICa was less pHi sensitive than late ICa (170 ms after depolarization to 0 mV). pHi-effects on single Ca2+ channel currents were studied with 110 mM BaCl2 as the charge carrier (22 degrees C, pHo 7.4). In cell-attached patches pHi was changed by extracellular NH4Cl or through the opened cell. In inside-out patches pHi was controlled through the bath. Independent of the method used the following results were obtained: (a) Single channel conductance (24 pS) and life time of the open state were not influenced by pHi (between pHi 6 and 8.4). (b) Alkaline pHi increased and acidic pHi reduced the channel availability (frequency of nonblank sweeps). (c) Alkaline pHi increased and acidic pHi reduced the frequency of late channel re-openings. The effects are discussed in terms of a deprotonation (protonation) of cytosolic binding sites that favor (prevent) the shift of the channels from a sleepy to an available state. Changes of bath pHo mimicked the pHi effects within 20 s, suggesting that protons can rapidly permeate through the surface membrane of vascular smooth muscle cells. The role of pHi in Ca2+ homeostases and vasotonus is discussed.

Calcium channel current of vascular smooth muscle cells: extracellular protons modulate gating and single channel conductance.

Modulation of L-type Ca2+ channel current by extracellular pH (pHo) was studied in vascular smooth muscle cells from bovine pial and porcine coronary arteries. Relative to pH 7.4, alkaline pH reversibly increased and acidic pH reduced ICa. The efficacy of pHo in modulating ICa was reduced when the concentration of the charge carrier was elevated ([Ca2+]o or [Ba2+]o varied between 2 and 110 mM). Analysis of whole cell and single Ca2+ channel currents suggested that more acidic pHo values shift the voltage-dependent gating (approximately 15 mV per pH-unit) and reduce the single Ca2+ channel conductance gCa due to screening of negative surface charges. pHo effects on gCa depended on the pipette [Ba2+] ([Ba2+]p), pK*, the pH providing 50% of saturating conductance, increased with [Ba2+]p according to pK* = 2.7-2.log ([Ba2+]p) suggesting that protons and Ba2+ ions complete for a binding site that modulates gCa. The above mechanisms are discussed in respect to their importance for Ca2+ influx and vasotonus.

Native-type DHP-sensitive calcium channel currents are produced by cloned rat aortic smooth muscle and cardiac alpha 1 subunits expressed in Xenopus laevis oocytes and are regulated by alpha 2- and beta-subunits.

Native tissue-like L-type voltage-dependent calcium channels (L-VDCC's) were expressed by in vitro transcribed cRNA injection of rat aorta or rabbit cardiac alpha 1 subunit into Xenopus laevis oocytes. Co-injection of VSM-alpha 1 with the cloned skeletal muscle beta-subunit (SK-beta) of the L-type VDCC significantly increased the expressed peak current amplitude without significant changes in kinetics. Similar results were obtained by co-injection of cardiac alpha 1 (DSHT-alpha 1) the cloned skeletal alpha 2-subunit (SK-alpha 2) or with SK-beta. The oocytes co-expressing cRNA's retained L-type VDCC pharmacology.

Isoforms of alpha1E voltage-gated calcium channels in rat cerebellar granule cells--detection of major calcium channel alpha1-transcripts by reverse transcription-polymerase chain reaction.

In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca2+ channels have been amplified by reverse transcription-polymerase chain reaction and identified by sequencing of subcloned polymerase chain reaction products. In these neurons cultured for six to eight days in vitro, fragments of the three major transcripts alpha1C, alpha1A, and alpha1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca2+ current recordings from six to eight days in vitro granule cells show that most of the current is due to L-type (25%), P-type (33%) and R-type (30%) Ca2+ channels. These data support the correlation between alpha1A and P-type Ca2+ channels (G1) and between alpha1E and R-type channels (G2 and G3). By including specific primer pairs for alpha1E the complimentary DNA fragments of indicative regions of alpha1E isoforms are amplified corresponding to the three most variable regions of alpha1E, the 5'-end, the II/III-loop, and the central part of the 3'-end. Although the complementary DNA fragments of the 5'-end of rat alpha1E yield a uniform reverse transcription-polymerase chain reaction product, its structure is unusual in the sense that it is longer than in the cloned rat alpha1E complementary DNA. It corresponds to the alpha1E isoform reported for mouse and human brain and is also expressed in cerebellum and cerebrum of rat brain as the major or maybe even the only variant of alpha1E. While fragments of a new rat alpha1E isoform are amplified from the 5'-end, three known fragments of the II/III-loop and two known isoforms homologue to the 3'-coding region are detected, which in the last case are discriminated by a 129 base pair insertion. The shift of the alpha1E expression from a pattern seen in cerebellum (alpha1Ee) to a pattern identified in other regions of the brain (alpha1E-3) is discussed. These data show that: (i) alpha1E is expressed in rat brain as a structural homologue to the mouse and human alpha1E; and (ii) rat cerebellar granule cells in primary culture express a set of alpha1E isoforms, containing two different sized carboxy termini. Since no new transcripts of high-voltage-activated Ca2+ channels genes are identified using degenerate oligonucleotide primer pairs, the two isoforms differentiated by the 129 base pair insertion might correspond to the two R-type channels, G2 and G3, characterized in these neurons. Functional studies including recombinant cells with the different proposed isoforms should provide more evidence for this conclusion.

Immunohistochemical detection of alpha1E voltage-gated Ca(2+) channel isoforms in cerebellum, INS-1 cells, and neuroendocrine cells of the digestive system.

Polyclonal antibodies were raised against a common and a specific epitope present only in longer alpha1E isoforms of voltage-gated Ca(2+) channels, yielding an "anti-E-com" and an "anti-E-spec" serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an alpha1E splice variant with a longer carboxy terminus, the so-called alpha1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter alpha1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer alpha1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas, alpha1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans. alpha1E therefore appears to be a common voltage-gated Ca(2+) channel linked to neuroendocrine and related systems of the body.

The dihydropyridine niguldipine modulates calcium and potassium currents in vascular smooth muscle cells.

1. Vascular smooth muscle cells were isolated from the portal vein and from pial vessels of the cow. They were voltage-clamped with a single patch electrode technique (whole cell recording) in order to analyse the effects of niguldipine on ionic membrane currents. Due to adsorption of niguldipine to plastic and glass, the effective concentrations are lower than the nominal concentrations by a factor of about 3. 2. Niguldipine reduced Ca-currents (ICa of the L-type, voltage operated) at nominal concentrations greater than 0.1 microM up to a complete block at 1 microM (50% block at 0.4 microM). Nominal concentrations between 50 and 200 nM facilitated ICa ('Ca-agonistic effect'). The Ca-agonistic effects of niguldipine showed modest use- but strong voltage-dependence. 3. Niguldipine increased the outward currents at nominal concentrations greater than 10 nM. The extra outward currents reversed at -85 mV, the result suggesting that niguldipine had increased potassium currents, IK. Maximal facilitation of IK by niguldipine was about 400% and was obtained at 1 microM, half-maximal facilitation was obtained with a nominal concentration of 20 nM. 4. Both reduction of ICa and facilitation of IK may contribute to vasodilatation by niguldipine. Due to its greater sensitivity, the effects on IK may dominate.

Carbon monoxide inhibits depolarization-induced Ca rise and increases cyclic GMP in visceral smooth muscle cells.

Monocytes were isolated from the urinary bladder of the guinea-pig. By means of the voltage clamp technique, whole cells were depolarized from -65 to +10 mV in order to increase the intracellular calcium concentration [Ca2+]i and to monitor this increase by means of the calcium activated potassium current IK.Ca. Superfusion of the cells with carbon monoxide-containing solutions for 2 min inhibited the signal to about 50% of the control suggesting depression of the depolarization-induced increase in [Ca2+]i. The CO-mediated inhibition of IK.Ca was partially reversed by wash-off of CO; flashes of high light intensity accelerated the rate of recovery. Sodium nitroprusside (0.01-1 mM) depressed the depolarization-induced increase in [Ca2+]i similar to CO. In multicellular preparations of the urinary bladder, CO-containing media were shown to increase the cGMP concentration by a factor of 2 in the absence and by a factor of 3 in the presence of 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). According to our previous work, CO binds to and activates soluble guanylate cyclase [Brüne B and Ullrich V, Mol Pharmacol 32: 497-504, 1987; Utz J and Ullrich V, Naunyn Schmiedebergs Arch Pharmacol 337 (Suppl): 299, 1988] and the rise in cGMP could thus effect [Ca2+]i by still unknown mechanisms.

Tiapamil reduces the calcium inward current of isolated smooth muscle cells. Dependence on holding potential and pulse frequency.

The calcium currents (ICa) of isolated smooth muscle cells (urinary bladder of the guinea-pig) were analyzed at 35 degrees C and 3.6 mM [Ca]0. The whole cells were voltage-clamped with a single patch electrode which was filled with 150 mM CsCl in order to block potassium outward currents. Tiapamil reduced ICa at concentrations between 1 microM (threshold) and 0.5 mM (complete block). Administration of 10 microM tiapamil at rest reduced ICa by 10% ('initial block'). Repetitive depolarizations (140 ms long pulses to -5 mV, applied at 1 Hz) reduced ICa further in a beat-to-beat fashion. This 'conditioned block' developed with a faster time course and to a greater extent when the stimulation frequency was increased and when the holding potentials were set to more positive levels. Reduction of ICa by tiapamil was facilitated by more positive and attenuated by more negative holding potentials. The influence of holding potential and pulse frequency on the tiapamil effect is discussed in terms of the 'modulated receptor hypothesis'.

Myocytes isolated from porcine coronary arteries: reduction of currents through L-type Ca-channels by verapamil-type Ca-antagonists.

Myocytes were enzymatically isolated from large epicardial arteries of the pig. In the cell attached configuration, we studied currents through L-type Ca-channels. At 22 degrees C, open channel conductance was 9 pS with 110 mM Ca2+ and 24 pS with 110 mM Ba2+ as charge carrier. According to the life time of the open state, 2 'modes' of gating are distinguished; mode 1 contributed time constants shorter than 1 ms, mode 2 those longer than 6 ms to the open time distribution. Mode 2 openings appeared spontaneously, more frequently with Ba2+ than with Ca2+ as charge carrier. The Ca-agonist Bay K 8644 (0.5 microM) facilitated the appearance of mode 2. Bath application of the phenylalkylamine D600 (1 microM) did not change the gating modes, but it reduced the channel openness by increasing the percentage of blank records. With whole cell recordings, we studied reduction of ICa by 1 microM D 600 at 3.6 mM [Ca2+] and 35 degrees C. At a holding potential of -45 mV, D 600 induced an 'initial block' of 35% (10% at -65 mV). Upon repetitive 1 Hz pulsing (170 ms to 0 mV) an additional, 'use-dependent' block developed with time. More negative holding potentials attenuated reduction of ICa by D 600, hyperpolarizations to -100 mV had an 'unblocking' effect. In regard to reduction of ICa, we compared the partially uncharged D 600 (membrane permeable) with the completely charged compound D 890 (membrane impermeable). When applied with the bath, 1 or 10 microM D 600 reduced ICa dose-dependently whereas D 890 was ineffective. When D 890 was applied via the patch electrode to the cytosol, it reduced ICa. We discuss that D 600 enters the cell in the uncharged lipid soluble form and reaches form the inside its receptor associated with the Ca-channel.

Intracellular calcium ions activate a low-conductance chloride channel in smooth-muscle cells isolated from human mesenteric artery.

Calcium-activated chloride currents were studied by the patch-clamp technique in vascular smooth muscle cells (VSMC) isolated from human mesenteric arteries. Bath application of 20 mM caffeine caused the cell membrane to depolarize by a calcium-activated inward current that peaked to -654 +/- 230 pA (holding potential -50 mV). Cell-attached, at the same time inwardly directed single-channel currents were detected with an amplitude of -0.22 pA. In open-cell-attached patches channel activity was triggered by elevating [Ca2+]i to 10 microM. At -60 mV the mean amplitude of the current was -0.24 pA and the mean open time of the channels was 28 ms. Plotting the amplitude of the current versus the test potential yielded a single-channel conductance of 2.8 +/- 0.5 pS. The currents disappeared when [Cl-] was reduced from 150 mM to 5 mM at the cytosolic side of the inside-out patch at a holding potential of -60 mV (calculated reversal potential -58 mV) suggesting that the calcium-activated current was a chloride current. This suggests that, in human mesenteric VSMC, elevation of [Ca2+]i activates a low-conductance chloride channel, which may mediate the agonist-induced depolarization of the cell membrane.

Glycocalyx is not required for show inward calcium current in isolated rat heart myocytes.

The importance of the slow inward calcium current (Isi) in the excitation-contration coupling process of cardiac muscle is well documented. The current can be attributed mainly to a calcium translocation from the extracellular space into the cell or a subsarcolemmal compartment of it. Isi has been suggested to have its source in and to be controlled by the surface coat of the sarcolemma (glycocalyx). The glycocalyx is destroyed in myocytes dissociated from adult heart tissue with solutions containing low calcium, collagenase and hyaluronidase (Fig. 1). By comparing the Isi data obtained in isolated myocytes with those reported for trabeculae or papillary muscles, we have now obtained evidence suggesting that the glycocalyx is not important in the genesis of Isi.

Calmodulin antagonists depress calcium and potassium currents in ventricular and vascular myocytes.

Myocytes isolated from guinea pig ventricles or bovine portal veins were voltage clamped with a single patch electrode. The calmodulin antagonists (CaM-A) calmidazolium, trifluoperazine (TFP), and chlorpromazine acted as Ca antagonists; they reduced the calcium inward current ICa in a voltage- and use-dependent way. For ventricular myocytes, 50% effective concentration (EC50) of calmidazolium was 1 microM, and the EC50 for TFP was 2.5 microM. For vascular myocytes, these numbers were 0.3 and 1 microM, respectively. CaM-A moderately retarded the inactivation time course and shifted the ICa availability curve to more negative potentials. CaM-A were not selective Ca antagonists; other membrane currents such as sodium currents and inwardly and delayed potassium currents were reduced as well (EC50 between 5 and 10 microM). It is unlikely that the above effects require binding of CaM-A to Ca-calmodulin, since reduction of ICa or potassium current (IK) was not modified when 1) the cells were loaded with 100 microM exogenous calmodulin or 2) Ca ions were removed from the extra- and intracellular space. Instead, the unspecific reduction of membrane currents may result from a change in the lipids of the sarcolemma into which CaM-A partition and accumulate.

The electrophysiological properties of the isolated adult heart cell: an overview.

We review the 3 current components iNa, iCa and iK and present those data which have been recently obtained from single cells. We discuss how the new data compare with those from multicellular tissue, and what they contribute to our understanding of the cardiac action potential. For the reasons of space and clarity, some of the problems will be oversimplified and presented incompletely.

Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current.

The effect of endothelin (ET) on membrane potential and current was studied in myocytes isolated from porcine coronary or from human mesenteric arteries at 3.6 mM extracellular Ca2+ concentration and 37 degrees C. ET (1-100 nM) induced cell shortening and membrane depolarization from a resting potential of -50 mV to about -15 mV. Ca currents (ICa, L-type) were transiently reduced by ET. At -50 mV, ET induced an inward current that peaked within 2 s and fell within 10 s to a sustained level. The current could be enlarged by reducing bath extracellular Cl- ion concentration, but removal of extracellular Na+ ions had no effect. The voltage dependence suggests that the ET-induced current is a Cl current (ICl) at potentials negative to -30 mV; at more positive potentials K currents (IK,Ca) are superimposed. The effects of ET on ICa, ICl, IK,Ca and contraction were prevented by intracellular Ca chelators, suggesting a Ca-dependent activation mechanism. The ET effects were abolished by pretreatment with 20 mM caffeine or prior cell-dialysis with heparin [thought to block inositol triphosphate-induced sarcoplasmic reticular Ca release]. The results suggest that ET releases Ca from the SR through a phosphoinositol response and that the released Ca acts as second messenger in modulating the membrane currents.

ATP suppresses activity of Ca(2+)-activated K+ channels by Ca2+ chelation.

Ca(2+)-activated maxi K+ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pfügers Arch 417:517, 1990), channel activity (NPo) was stimulated by 3 microM [Ca2+]c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na2ATP. The following results suggest that suppression of NPo by Na2ATP is due to Ca2+ chelation and hence reduction of [Ca2+]c and reduced Ca2+ activation of the channel. The effect was absent when Mg ATP was used instead of Na2ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca2+]c was buffered with 10 mM HDTA (apparent pKCa 5.58) instead of EGTA (pKCa 6.8). A Ca(2+)-sensitive electrode system indicated that 1 mM Na2ATP reduced [Ca2+]c in 1 mM Ca-EGTA from 3 microM to 1.4 microM. Na2ATP, Na2GTP, Li4AMP-PNP and NaADP reduced measured [Ca2+]c in parallel with their suppression of NPo. After the Na2ATP-induced reduction of [Ca2+]c was re-adjusted by adding either CaCl2 or MgCl2, the effect of Na2ATP on NPo disappeared. In vivo, intracellular [Mg2+] exceeds free [ATP4-], hence ATP modulation of maxi K+ channels due to Ca2+ chelation is without biological relevance.

Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig).

Cells were isolated by incubating chunks of tissue from the urinary bladder of the guinea-pig in a high potassium, low chloride medium containing 0.2 mM calcium plus the enzymes collagenase and pronase. After isolation, the cells were superfused with a physiological salt solution (PSS) containing 150 mM NaCl, 3.6 mM CaCl2 and 5.4 mM KCl (35 degrees C). Patch electrodes filled with an isotonic KCl-solution were used for whole cell recordings. With a single electrode voltage clamp we measured a capacitance of 50 +/- 5 pF per cell, an input resistance of 200 +/- 25 kOhm X cm2 and a series resistance of 44 +/- 4 Ohm X cm2. The cells had resting potentials of -52 +/- 2 mV. They did not beat spontaneously but responded to stimuli with single action potentials (APs) which rose from the threshold (-38 mV) with a maximal rate of 6.5 +/- 1.8 V/s to an overshoot of 22 +/- 3 mV. The AP lasted for 36 +/- 4 ms (measured between threshold and -40 mV). Continuous cathodal current produced repetitive activity, a pacemaker depolarization followed the AP and preceded the next upstroke. Net membrane currents evoked by clamp steps to positive potentials were composed of an inward and an outward component. The inward component generating the upstroke of the AP was carried by Ca ions (iCa, Klöckner and Isenberg 1985). The repolarization resulted from a potassium outward current iK. Ca-channel blockers (5 mM NiCl2) reduced iK suggesting that (part of) iK was Ca-activated. iK rose within about 100 ms to a peak of 40-200 muA/cm2 from which it inactivated slowly and incompletely. The inactivating iK followed a bell-shaped voltage-dependence, the noninactivating iK an outwardly rectifying one. Both parts had similar steady state inactivation curves with a half maximal inactivation potential at -36 mV and a slope of 9 mV. Repolarization to -50 mV induced outward tail currents which reversed polarity at -85 mV (the calculated potassium equilibrium potential). The amplitude and the time course of the envelope of the tail currents varied in proportion to iK during the prestep. Thus, the tail current is suggested to reflect the turning off of a potassium conductance which had been activated during the prepulse. iK was largely reduced but not blocked by 20 or 150 mM tetraethylammonium (TEA). TEA did not significantly change the resting potential, but it prolonged the AP and facilitated upstroke and overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig).

Single smooth muscle cells isolated from the urinary bladder of the guinea-pig were studied at 35 degrees C in a solution composed of 150 mM NaCl, 3.6 mM CaCl2, 1.2 mM MgCl2, 5.4 mM KCl, 20 mM TEA-Cl, 5 mM glucose, 10 mM HEPES/NaOH (pH 7.4). Whole cells were clamped with a single patch electrode. The clamp settled a step from -65 to -5 mV within 260 microseconds, and afterwards the voltage inhomogeneities were less than 2 mV (measured at the cell edge with a second electrode). The calcium inward current iCa was dissected from net currents by blocking potassium outward currents by means of patch electrodes filled with 130 mM CsCl (Klöckner and Isenberg 1985 a). Pyruvate, succinate and oxalacetate in the patch electrode stabilized iCa and prevented its "run down". 140 ms long clamp steps from -65 to -5 mV evoked a net inward current which could be reversibly blocked by 5 mM NiCl2. The "Ni-sensitive" difference current iCa peaked within 2-4 ms to about 1 nA per cell. Afterwards it completely inactivated; the inactivation could be fitted with three exponentials (time constants of 4, 30, and 250 ms, respectively). The half decay time of 16 ms suggests that most of the inactivation resulted from the fast exponential process. The reference current in the presence of Ni was nearly time independent and almost zero; therefore, iCa could be approximated from the net inward current using the zero current as a reference line.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca-tolerant guinea-pig ventricular myocytes as isolated by pronase in the presence of 250 microM free calcium.

A new method to isolate adult cardiocytes with pronase in the presence of 250 microM free calcium was developed. Ultrastructural and electrophysiological properties of these cells were investigated. It is shown that by this method normal calcium-tolerant cells can be obtained.

Ca-antagonistic effects of adenosine in guinea pig atrial cells.

In atrial myocytes of the guinea pig, the effects of adenosine (Ado) and acetylcholine (ACh) on Ca currents (ICa) were investigated with the patch-electrode whole cell clamp technique. ICa was dissected from net currents by blocking K currents (IK) with intra- and extracellular Cs ions. ICa was considered as "basal" ICa, since it was not prestimulated by beta-agonists (isoproterenol). T-channel Ca currents were insensitive to Ado or ACh. The antagonism of L-channel Ca currents was maximal with 10 microM Ado or 3 microM ACh, which reduced basal ICa by 35%. From the concentration dependence, a dissociation constant (KD) value of 1.1 microM Ado and a Hill coefficient of -3 were obtained. Ado and ACh were not additive but saturative in reducing basal ICa. Reduction of basal ICa did not modify inactivation time-course, steady-state activation or inactivation, suggesting that Ado reduces the number of functional Ca channels. In myocytes with unblocked IK (KCl electrodes), 3 microM Ado (1 microM ACh) reduced ICa by 30% but increased IK by 300%. It is concluded that the K-agonistic rather than the Ca-antagonistic effect accounts for hyperpolarization as well as for most of the shortening of the action potential and the negative inotropy.