Calcium channel activation in vascular smooth muscle by BAY K 8644

BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate) and CGP 28 392 (ethyl-4(2-difluoromethoxyphenyl)-1,4,5,7-tetrahydro-2-methyl-5-++ +oxofuro- [3,4-b]pyridine-3-carboxylate) are closely related in structure to nifedipine and other 1,4-dihydropyridine Ca2+ channel antagonists. However, both BAY K 8644 and CGP 28 392 serve as activators of Ca2+ channels. In the rat tail artery, responses to BAY K 8644 are dependent upon Ca2+ext and prior stimulation by K+ or by the alpha-adrenoceptor agonists, phenylephrine and BHT 920 (6-allyl-2-amino-5,6,7,8,-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). Responses are blocked noncompetitively by the Ca2+ channel antagonists D-600 [-)-D-600 greater than (+)-D-600) and diltiazem, but competitively by nifedipine (pA2 = 8.27). This suggests that activator and inhibitor 1,4-dihydropyridines interact at the same site. BAY K 8644 potentiates K+ responses and Ca2+ responses in K+-depolarizing media. The leftward shift of the K+ dose--response curve produced by BAY K 8644 suggests that this ligand facilitates the voltage-dependent activation of the Ca2+ channel. The pA2 value for nifedipine antagonism of BAY K 8644 responses is significantly lower than that for nifedipine antagonism of Ca2+ responses in K+ (25-80 mM) depolarizing media (9.4-9.6), suggesting that the state of the channel may differ according to the activating stimulus.     

Phosphorylation shifts the time-dependence of cardiac Ca++ channel gating currents.

A general mechanism for the physiological regulation of the activity of voltage-dependent Na+, Ca++, K+, and Cl channels by neurotransmitters in a variety of excitable cell types may involve a final common pathway of a cyclic AMP-dependent phosphorylation of the channel protein. The functional correlates of channel phosphorylation are known to involve a change in the probability of opening, and a negative or positive shift in the voltage dependence for activation of the conductance. The voltage dependence for activation appears to be governed by the properties of the charge movement of the voltage-sensing moiety of the channel. This study of the gating charge movement of cardiac Ca++ channels has revealed that isoproterenol or cAMP (via a presumed phosphorylation of the channel) speeds the kinetics of the Ca++ channel gating charge movement. These results suggest that the changes in the kinetics and voltage dependence of the cardiac calcium currents produced by beta-adrenergic stimulation are initiated, in part, by parallel changes in the gating charge movement.     

Coupling of voltage-dependent gating and Ba++ block in the high- conductance, Ca++-activated K+ channel

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.     

Resolving the gating charge movement associated with late transitions in K channel activation.

We examined the late transitions in the activation sequence of potassium channels by analyzing gating currents of mutant Shaker IR channels and using the potassium channel blocker 4-aminopyridine (4AP). Gating currents were recorded from a double mutant of Shaker that was nonconducting (W434F mutation) and had the late gating transitions shifted to the right on the voltage axis (L382C mutation), thus separating the late transitions from the early ones. 4AP applied to the double mutant blocked the final transition and made possible novel observations of the isolated intermediate transitions, the ones that immediately precede the final opening of the channel. These transitions, which have not been well characterized previously, produce a distinct fast component in the gating current tails. Two intermediate transitions contribute to the fast component and carry 23% of the total gating charge. The effect of 4AP is well modeled as a selective block of the final gating transition, which opens the channel. The final transition contributes approximately 5% of the total gating charge.     

Calcium channel modulation by the dihydropyridine derivative Bay K 8644 in mammalian visceral smooth muscle cells

1. Modulation of Ca channels by the dihydropyridine Ca agonist Bay K 8644 in guinea-pig taenia coli smooth muscle cells was investigated using the patch clamp technique. 2. Single Ca channel activity was obtained from cell-attached patch recordings with the use of pipettes filled with 50 mM Ba. Bath application of the drug markedly increased the opening probability of Ca channels. 3. The effect was found to be due to an increase in the mean opening times of Ca channels. Due to this increase, the mean current reconstructed by averaging individual current trace responses was markedly increased in the presence of Bay K 8644.     

Voltage-dependent modulation of Ca channel current in heart cells by Bay K8644

We have investigated the voltage-dependent effects of the dihydropyridine Bay K8644 on Ca channel currents in calf Purkinje fibers and enzymatically dispersed rat ventricular myocytes. Bay K8644 increases the apparent rate of inactivation of these currents, measured during depolarizing voltage pulses, and shifts both channel activation and inactivation in the hyperpolarizing direction. Consequently, currents measured after hyperpolarizing conditioning pulses are larger in the presence of drug compared with control conditions, but are smaller than control if they are measured after positive conditioning pulses. Most of our experimental observations on macroscopic currents can be explained by a single drug-induced change in one rate constant of a simple kinetic model. The rate constant change is consistent with results obtained by others with single channel recordings.     

Differential modulation of cardiac Ca2+ channel gating by beta-subunits

To investigate the mechanisms that increase ionic currents when Ca(2+) channels' alpha(1) subunits are co-expressed with the beta-subunits, we compared channel activity of Ca(V)1.2 (alpha(1C)) co-expressed with beta(1a) and beta(2a) in Xenopus oocytes. Normalized by charge movement, ionic currents were near threefold larger with beta(2a) than with beta(1a). At the single-channel level, the open probability (P(o)) was over threefold larger with beta(2a), and traces with high P(o) were more frequent. Among traces with P(o) > 0.1, the mean duration of burst of openings (MBD) were nearly twice as long for alpha(1C)beta(2a) (15.1 +/- 0.7 ms) than for alpha(1C)beta(1a) (8.4 +/- 0.5 ms). Contribution of endogenous beta(3xo) was ruled out by comparing MBDs with alpha(1C)-cRNA alone (4.7 +/- 0.1 ms) with beta(3xo) (14.3 +/- 1.1 ms), and with beta(1b) (8.2 +/- 0.5 ms). Open-channel current amplitude distributions were indistinguishable for alpha(1C)beta(1a) and alpha(1C)beta(2a), indicating that opening and closing kinetics are similar with both subunits. Simulations with constant opening and closing rates reproduced the microscopic kinetics accurately, and therefore we conclude that the conformational change-limiting MBD is differentially regulated by the beta-subunits and contributes to the larger ionic currents associated with beta(2a), whereas closing and opening rates do not change, which should reflect the activity of a separate gate.     

In vitro secretion of calcitonin from a rat C cell line: effect of repetitive stimulation with the calcium channel agonist BAY K 8644.

Calcium and BAY K 8644 acutely stimulate calcitonin secretion by influx of extracellular calcium (Ca) through voltage-dependent calcium channels, leading to an increase in cytosolic free Ca. Repetitive exposure to BAY K 8644 (10(-6) M) resulted in an increase in calcitonin (CT) secretion in the rat C-cell line (rMTC 6-23) lasting 9 hours, in comparison to that of 3 mM Ca2+ which lasted 6 hours. Equimolar concentration of nifedipine did not inhibit the stimulatory effect of BAY K 8644 as compared to the nifedipine only group. The decrease in stimulated CT secretion during long-term exposure to BAY K 8644 is due to desensitization of cells which may be attributed to down-regulation of dihydropyridine receptors. After 12 h exposures to 3 mM Ca2+ alone, BAY K 8644 (10(-6) M) alone or in combination with nifedipine (10(-6) M), CT content decreased below the control level, indicating a decrease in synthesis. Overall cellular protein content was not affected by the test agents. Repetitive exposure of C-cells to BAY K 8644 revealed a desensitization of the stimulatory effect on CT secretion and a decrease in CT cell content.     

The beta subunit controls the gating and dihydropyridine sensitivity of the skeletal muscle Ca2+ channel.

The skeletal muscle (SKM) L-type Ca2+ channel is composed of a central subunit designated alpha 1, which contains the pore and the dihydropyridine (DHP) binding domains and three associated subunits, alpha 2/delta, beta, and gamma, which influence the activity of the SKM alpha 1. Coexpression of SKM alpha 1 and SKM beta in stably transfected mouse L cells results in a dramatic increase in DHP binding accompanied by fast gated Ba2+ currents. We report here that this "SKM alpha 1 beta-related phenotype" can be converted upon intracellular trypsin treatment into a slowly inactivating, DHP sensitive "SKM alpha 1 phenotype." These observations indicate that current amplitude, fast inactivation, and DHP sensitivity are modulated by an interaction of SKM alpha 1 and SKM beta on the internal side of the membrane.     

The calcium channel agonist, Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues

Effects of a novel slow channel activator, Bay K-8644 (Bay K), were studied on slow action potential (APs) in young and old embryonic chick hearts, and on its antagonism of the effects of diacetyl monoxime (DAM). The slow APs of young hearts are mediated by slow Na+ channels, whereas those of old hearts are mediated by slow Ca2+ channels. In slow APs of old (13-18 days old) embryonic chick hearts superfused with a high (22 mM) K+ solution, Bay K (10-6 M) gradually increased the amplitude, maximum rate of rise (Vmax), and duration of the slow APs. The actions of Bay K persisted for a long time (greater than 30 min) after washout of the drug. DAM (10 mM) depressed the Vmax, duration and amplitude of the slow APs. Some of the changes in slow AP parameters produced by DAM, e.g., Vmax decrease, were antagonized by the addition of Bay K (10(-6) M). In 3-day-old embryonic chick hearts. Bay K potentiated the slow APs and DAM depressed them; Bay K antagonized these effects of DAM. Thus, the actions of Bay K and DAM are likely to be produced, respectively, via the activation and depression of slow Ca2+ channels in old embryonic chick hearts. In addition, the drugs seem to influence slow Na+ channels found in young embryonic chick hearts.     

Ca2(+)-channel agonist BAY K8644 mimics 1,25(OH)2-vitamin D3 rapid enhancement of Ca2+ transport in chick perfused duodenum.

To further understand the molecular mechanism by which 1,25(OH)2-vitamin D3 [1,25(OH2D3] rapidly stimulates intestinal calcium transport (termed "transcaltachia"), the effect of the calcium channel agonist BAY K8644 was studied in vascularly perfused duodenal loops from normal, vitamin D-replete chicks. BAY K8644, 2 mu M, was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25(OH)2D3. The sterol and the Ca2+ channel agonist both increased 45Ca2+ transport 70% above control values within 2 min and 200% after 30 min of vascular perfusion. The effect of the Ca2+ channel agonist was dose dependent. Also, 1,25(OH)2D3-enhanced transcaltachia was abolished by the calcium channel blocker nifedipine. Collectively, these results suggest the involvement of 1,25(OH)2D3 in the activation of basal lateral membrane Ca2+ channels as an early effect in the transcaltachic response.     

Partitioning and location of Bay K 8644, 1,4-dihydropyridine calcium channel agonist, in model and biological membranes.

Several lines of evidence suggest that nonspecific drug interaction with the lipid bilayer plays an important role in subsequent recognition and binding to specific receptor sites in the membrane. The interaction of Bay K 8644, a 1,4-dihydropyridine (DHP) calcium channel agonist, with model and biological membranes was examined at the molecular level using small angle x-ray diffraction. Nonspecific drug partitioning into the membrane was examined by radiochemical assay. Nonspecific binding characteristics of [3H] Bay K 8644 were determined in both dipalmitoyl phosphatidylcholine (DPPC) vesicles above and below their thermal phase transition (Tm) and rabbit skeletal muscle light sarcoplasmic reticulum (LSR). In DPPC, the partition coefficient, Kp, was 14,000 above the Tm (55 degrees C) versus 160 in the gel phase (2 degrees C). The Kp determined in LSR membranes was 10,700. These values for both DPPC and LSR membranes can be compared with Kp = 290 in the traditional octanol/buffer system. Using small-angle x-ray diffraction, the equilibrium position of the electron-dense trifluoromethyl group of Bay K 8644 in DPPC (above Tm) and purified cardiac sarcolemmal (CSL) lipid bilayers was determined to be consistently located within the region of the first few methylene segments of the fatty acyl chains of these membranes. This position is similar to that observed for the DHP calcium channel antagonists nimodipine and Bay P 8857. We suggest this particular membrane location defines a region of local drug concentration and plane for lateral diffusion to a common receptor site. Below the DPPC membrane Tm, Bay K 8644 was shown to be excluded from this energetically favored position into the interbilayer water space. Heating the DPPC bilayer above the Tm (55 degrees C) showed that this exclusion was reversible and indicates that drug-membrane interaction is dependent on the bilayer physical state. The absence of any specific protein binding sites in these systems allows us to ascertain the potentially important role that the bulk lipid phase may play in the molecular mechanism of DHP binding to the specific receptor site associated with the calcium channel.     

Potentiation of the cardiac L-type Ca(2+) channel (alpha(1C)) by dihydropyridine agonist and strong depolarization occur via distinct mechanisms.

A defining property of L-type Ca(2+) channels is their potentiation by both 1,4-dihydropyridine agonists and strong depolarization. In contrast, non-L-type channels are potentiated by neither agonist nor depolarization, suggesting that these two processes may by linked. In this study, we have tested whether the mechanisms of agonist- and depolarization-induced potentiation in the cardiac L-type channel (alpha(1C)) are linked. We found that the mutant L-type channel GFP-alpha(1C)(TQ-->YM), bearing the mutations T1066Y and Q1070M, was able to undergo depolarization-induced potentiation but not potentiation by agonist. Conversely, the chimeric channel GFP-CACC was potentiated by agonist but not by strong depolarization. These data indicate that the mechanisms of agonist- and depolarization-induced potentiation of alpha(1C) are distinct. Since neither GFP-CACC nor GFP-CCAA was potentiated significantly by depolarization, no single repeat of alpha(1C) appears to be responsible for depolarization-induced potentiation. Surprisingly, GFP-CACC displayed a low estimated open probability similar to that of the alpha(1C), but could not support depolarization-induced potentiation, demonstrating that a relatively low open probability alone is not sufficient for depolarization-induced potentiation to occur. Thus, depolarization-induced potentiation may be a global channel property requiring participation from all four homologous repeats.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Reduced effects of BAY K 8644 on L-type Ca2+ current in failing human cardiac myocytes are related to abnormal adrenergic regulation

Abnormal L-type Ca(2+) channel (LTCC, also named Cav1.2) density and regulation are important contributors to depressed contractility in failing hearts. The LTCC agonist BAY K 8644 (BAY K) has reduced inotropic effects on failing myocardium. We hypothesized that BAY K effects on the LTCC current (I(CaL)) in failing myocytes would be reduced because of increased basal activity. Since support of the failing heart with a left ventricular assist device (LVAD) improves contractility and adrenergic responses, we further hypothesized that BAY K effects on I(CaL) would be restored in LVAD-supported failing hearts. We tested our hypotheses in human ventricular myocytes (HVMs) isolated from nonfailing (NF), failing (F), and LVAD-supported failing hearts. We found that 1) BAY K had smaller effects on I(CaL) in F HVMs compared with NF HVMs; 2) BAY K had diminished effects on I(CaL) in NF HVM pretreated with isoproterenol (Iso) or dibutyryl cyclic AMP (DBcAMP); 3) BAY K effects on I(CaL) in F HVMs pretreated with acetylcholine (ACh) were normalized; 4) Iso had no effect on NF HVMs pretreated with BAY K; 5) BAY K effects on I(CaL) in LVAD HVMs were similar to those in NF HVMs; 6) BAY K effects were reduced in LVAD HVMs pretreated with Iso or DBcAMP; 7) Iso had no effect on I(CaL) in LVAD HVMs pretreated with BAY K. Collectively, these results suggest that the decreased BAY K effects on LTCC in F HVMs are caused by increased basal channel activity, which should contribute to abnormal contractility reserve.     

Revisiting the role of Ca2+ in Shaker K+ channel gating

Shaker K+ channels were expressed in outside-out macropatches excised from Xenopus oocytes, and the effects on gating of removal of extracellular Ca2+ were examined in the complete absence of intracellular divalent cations. Removal of extracellular Ca2+ by perfusion with EDTA-containing solution caused a small negative shift in the channel's voltage-activation curve and led to an increased nonselective leak, but did not otherwise alter or disrupt the channels. The results contradict the proposal that Ca2+ is an essential component required for maintenance of ion selectivity and proper gating of Kv-type K+ channels. The large nonselective leak in Ca2+-free conditions was found to be a patch-seal phenomenon related to F- ion in the recording pipette.     

Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltage-dependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating models of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers

Summary A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K⁺ concentration that could not be described by simple Michaelis-Menten saturation. At low K⁺ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4m KCl. At the same Ca²⁺ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K⁺ and Ca²⁺ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K⁺-conduction and Ca²⁺-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 Å from the surface.     

Ca2+-dependent gating mechanisms for dSlo, a large-conductance Ca2+-activated K+ (BK) channel.

The Ca2+-dependent gating mechanism of cloned BK channels from Drosophila (dSlo) was studied. Both a natural variant (A1/C2/E1/G3/IO) and a mutant (S942A) were expressed in Xenopus oocytes, and single-channel currents were recorded from excised patches of membrane. Stability plots were used to define stable segments of data. Unlike native BK channels from rat skeletal muscle in which increasing internal Ca2+ concentration (Cai2+) in the range of 5 to 30 microM increases mean open time, increasing Cai2+ in this range for dSlo had little effect on mean open time. However, further increases in Cai2+ to 300 or 3000 microM then typically increased dSlo mean open time. Kinetic schemes for the observed Ca2+-dependent gating kinetics of dSlo were evaluated by fitting two-dimensional dwell-time distributions using maximum likelihood techniques and by comparing observed dependency plots with those predicted by the models. Previously described kinetic schemes that largely account for the Ca2+-dependent kinetics of native BK channels from rat skeletal muscle did not adequately describe the Ca2+ dependence of dSlo. An expanded version of these schemes which, in addition to the Ca2+-activation steps, permitted a Ca2+-facilitated transition from each open state to a closed state, could approximate the Ca2+-dependent kinetics of dSlo, suggesting that Ca2+ may exert dual effects on gating.