Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca²⁺-dependent inactivation (CDI) of high-voltage activated (HVA) Ca²⁺ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca²⁺-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34±9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31±3%), a low concentration (3 µM) of parvalbumin (20±2%) and calbindin_D28K (24±4%) significantly reduced CDI. Subtype-specific Ca²⁺ channel blockers indicated that L-type, but not N-type Ca²⁺ channels are governed by CDI and modulated by Ca²⁺-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca²⁺-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22±2%; calmodulin binding domain, 17±1%) and cytoskeleton stabilizers (taxol, 23±5%; phalloidin, 15±3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca²⁺ channels in thalamic physiology.     

Single Ca channel currents in mouse pancreatic B-cells

Barium currents flowing through single Ca²⁺ channels were recorded from outside-out patches isolated from mouse pancreatic B-cells. Only one type of Ca²⁺ channel was observed. In 110 mM Ba²⁺, the single channel conductance was 24 pS (at negative membrane potentials) and the current amplitude at 0 mV was–0.7 pA. Channel openings were activated by depolarisations more positive than –30 mV and showed little inactivation during 200 ms pulses. Open times were increased by BAY K 8644 an decreased by micromolar Cd²⁺. Channel activity was subject to rundown in excised patches and little activity remained after 10 min. These properties resemble those of L-type Ca²⁺ channels in other tissues. It is suggested that this Ca²⁺ channel participates in the generation of the B-cell action potential and mediates the increase in Ca²⁺ influx required for insulin secretion.     

Single Ca2+-activated K+ channels in excised membrane patches of hamster oocytes

The properties of the Ca²⁺-activated K⁺ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca²⁺-activated K⁺ channel which has the following characteristics: (1) single-channel conductance of 40–85 pS for outward currents in symmetrical K⁺ (150 mM) solutions, (2) inward currents of smaller conductance (10–50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K⁺ solutions, (3) channel activity dependent on the internal concentration of free Ca⁺ and the membrane potential, (4) modification of the channel activity by internal adenosine 5 diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.     

Blocking actions of Ca2+ antagonists on the Ca2+ channels in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists, verapamil, nicardipine and diltiazem, were investigated on the Ca²⁺ inward current in the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) obtained from the longitudinal muscle layer of the rabbit ileum, by enzymatic dispersion. All Ca²⁺ antagonists inhibited the inward current, in a dose-dependent manner. The ID₅₀ value on the maximum amplitude of the inward current of nicardipine was 24 nM, and this value was roughly 50 times lower than values obtained with verapamil and diltiazem, when the inward current was provoked by 0 mV command pulse from the holding potential of –60 mV. Lowering the holding potential to –80 mV shifted the dose-response curve to the right. When depolarizing pulses (100 ms, stepped up to 0 mV from –60 mV or –80 mV) were applied every 20 s, the peak amplitude of the inward current remained unchanged, but nicardipine immediately, and diltiazem and verapamil slowly reduced the peak amplitude. These slow inhibitions by the latter two drugs depended on the frequency or number of stimulations. Nicardipine but not diltiazem and verapamil shifted the voltage-dependent inactivation curve to the left (3 s duration of the conditioning pulse). However, with a longer conditioning pulse (10 s) verapamil and diltiazem shifted the voltage-dependent inactivation curves to the left. Therefore, the inhibitory actions of these Ca²⁺ antagonists differ. Namely, diltiazem and verapamil inhibit the Ca²⁺ channels, mainly in a frequency-or use-dependent manner while nicardipine does so in a voltage-dependent manner.     

Single Ca channel currents in mammalian visceral smooth muscle cells

Recordings of single Ca channel currents in mammalian visceral smooth muscle cells were obtained using patch clamp techniques. Smooth muscle cells from guinea-pig taenia coli were prepared by enzymatic dispersion using 0.3% collagenase. The recordings were obtained from cell-attached membrane patches of isolated cells with a pipette filled with isotonic 50 mM Ba²⁺. When the membrane patch was depolarized, brief inward current pulses of unitary size and small amplitude were observed. The amplitude of these single channel currents decreased linearly with increasing depolarization in a voltage range from –20 mV to +50 mV about the resting potential. The slope conductance was estimated to be about 30 pS. The mean current reconstructed by averaging individual current responses showed kinetic behaviour with a rapid activation and a slower inactivation process similar to the macroscopic Ca²⁺ current observed in strips of guinea-pig taenia coli. The present study suggests that the inward current pulses of unitary size induced by voltage-clamp pulses were due to Ba ions passing through a single voltage dependent Ca channel.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

Ca2+ channel Ca2+-dependent inactivation in a mammalian central neuron involves the cytoskeleton

Ca²⁺ channel inactivation was investigated in acutely isolated hippocampal pyramidal neurons from adult rats and found to have a component dependent on intracellular Ca²⁺. Ca²⁺-dependent inactivation was identified as the additional inactivation of channel current observed when Ca²⁺ replaced Ba²⁺ as the current carrying ion, and was found to be an independent process from that of Ba²⁺ current inactivation based on three lines of evidence: (1) no correlation between Ca²⁺-dependent inactivation and Ba²⁺ current inactivation was found, (2) only Ca²⁺-dependent inactivation was reduced by intracellular application of Ca²⁺ chelators, and (3) only Ca²⁺-dependent inactivation was sensitive to compounds which alter the cytoskeleton. Drugs which stabilize (taxol and phalloidin) and destabilize (colchicine and cytochalasin B) the cytoskeleton altered the development and recovery from Ca²⁺-dependent inactivation, indicating that the neuronal cytoskeleton may mediate Ca²⁺ channel sensitivity to intracellular Ca²⁺. Ca²⁺-dependent inactivation was not associated with a particular subset of Ca²⁺ channels, suggesting that all Ca²⁺ channels in these neurons are inactivated by intracellular Ca²⁺.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

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.     

Single channel activity associated with the calcium dependent outward current inHelix pomatia

A recently improved version of the extracellular patch clamp technique (9, 13) was used to record currents from microscopic membrane areas of Helix neurons with predominant Ca²⁺ dependent outward currents. Current fluctuations in the patches consisted mainly of frequently interrupted, one-sided steps indicating discrete open-closed state changes of single channels with an ohmic conductance of approximately 19 pS. Frequency of occurrence of the elementary events compares with amplitudes of macroscopic currents during depolarizing voltage steps of varied amplitude. Average delays in appearance of the events vary in line with delayed time courses of the cell's outward current.     

Effect of adrenergic agonists on Ca2+-channel currents in single vascular smooth muscle cells

Ca²⁺-channel currents have been measured in enzymatically dispersed single smooth muscle cells of the rabbit ear artery using the whole-cell patch clamp technique. Inward currents were elicited by depolarizing test pulses from a holding potential of–50 mV. These currents were activated from–30 mV onward and reached full activation around 0 mV. -Adrenergic agonists did not affect the background current measured at the holding potential, but markedly reduced the peak amplitude of the voltageactivated Ca²⁺-channel currents. This -adrenergic inhibition also occurred in cells which were internally perfused with solutions containing either 10 M cAMP, 10M cGMP or 0.1 mM GTP, but became irreversible when the pipette solution contained a non-hydrolyzable GTP-analog. The action of -agonists on the voltage-activated Ca²⁺-channel currents was variable, and ranged from no effect at all to a 50% reduction of the current. It is concluded that -agonists do not open receptor-operated Ca²⁺-channels in these smooth muscle cells. The inhibition of the voltageactivated Ca²⁺-currents does not seem to be mediated through changes in cyclic nucleotide levels, but might be mediated through G-proteins. Its physiological relevance remains however unclear. The action of -agonists is consistent with their relaxing effect, but the reason for the nonuniform response has not been elucidated.     

Evidence for contribution of Ca2+ storage sites on unitary K+ channel currents in inside-out membrane of rabbit portal vein

While making use of the inside-out membrane patch, we examined the effects of caffeine and heparin on unitary currents of the large conductance Ca²⁺ -dependent K⁺ (maxi-K⁺) channel in the rabbit portal vein. About half of the inside-out membranes we used contained a functional Ca²⁺ -store site which facilitated modification of the maxi-K⁺ channel.When high-K⁺ solution containing 0.05mM EGTA was superfused in the bath, simultaneous openings of more than 20 maxi-K⁺ channels were observed in 39 of 83 patch membranes, and multi-channel opening appeared periodically or continuously at the holding potential of – 10mV. Most channel activities of these patch membranes were inhibited by caffeine or heparin, and some heparin-insensitive channel activities were inhibited by caffeine. The remaining patch membranes (44 out of 83) showed low activity of the maxi-K⁺ channel, and neither caffeine nor heparin modified channel activity.Therefore, in our experimental set-up, half the number of excised patch membranes contained a Ca²⁺ store site. Most Ca²⁺ store sites have inositol 1,4,5-trisphosphate (InsP₃)-activated Ca²⁺ release (IACR) and caffeine-activated Ca²⁺ release (CACR) channels and few lack the IACR channel. The mechanisms of activation of the maxi-K⁺ channel in relation to release of Ca²⁺ from the store sites can be examined in detail using the approaches we have described.     

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.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

Increased Ca2+ channel currents in cerebellar Purkinje cells of the ataxic groggy rat

The ataxic groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats. Recent genetic analysis has identified the missense (M251K) mutation in the alpha(1) subunit of the Ca(V)2.1 (P/Q-type) voltage-dependent Ca(2+) channel gene (Cacna1a) of GRY rat. In this study, we found that high-voltage-activated (HVA) Ca(2+) channel currents in acutely dissociated Purkinje cells of GRY rats showed increased (not decreased) current density and depolarizing shift of the activation and inactivation curves compared with those of normal Wistar rats. In contrast low-voltage-activated (LVA) Ca(2+) channel currents of GRY rats showed no significant changes. These results suggest that functional alteration of Ca(2+) channel currents in cerebellar Purkinje cells of GRY rats is attributed to the change of HVA Ca(2+) channel currents, and that increased HVA Ca(2+) channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.     

Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.     

D600 blocks the Ca2+ channel from the outer surface of smooth muscle cell membrane of the rabbit intestine and portal vein

The voltage dependent Ca²⁺ inward current in single smooth muscle cells dispersed from the longitudinal muscle layer of the rabbit ileum and rabbit portal vein was recorded using the whole-cell voltage clamp technique. D600 added to the bathing solution inhibited the Ca²⁺ current, while the intracellular perfusion of this agent did not reduce the amplitude of this current. Thus, D600 probably acts from the outer surface of the membrane. The nature of the Ca²⁺ channel in smooth muscle cells seems to differ from that in cardiac muscle cells.     

A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel

The inactivation of voltage-gated L-type Ca(2+) channels (Ca(V)1) regulates Ca(2+) entry and controls intracellular Ca(2+) levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca(V)1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Ca(v)1.2, alpha(1)1.2 and alpha(1)1.2 DeltaN60-Delta1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca(V)1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca(2+) or Ba(2+) and was not altered by the beta2A subunit. Missing from the fast inactivating neuronal P/Q (Ca(V)2.1)-, N (Ca(V)2.2)- or R (Ca(V)2.3)-type channels and modulating Ca(V)1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca(2+) channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca(V)1.2 specific built-in "stopper." In this model, Ca(V)1 accomplishes a large Ca(2+) influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.     

Reciprocal effects of Ca2+ and Mg-ATP on the ‘run-down’ of the K+ channels in opossum kidney cells

Using the patch clamp technique, we identified an inwardly rectifying K⁺ channel in the membrane of opossum kidney cells. The single channel conductance was about 90 pS for inward currents and 30 pS for outward currents under a symmetrical high-K⁺ condition. The activity of the channel was found to decrease with time during recording from inside-out patches. In the solution with submicromolar Ca²⁺, the activity disappeared within 4–20 min. Intracellular Ca²⁺ promoted the run-down of the channel activity at 0.1–1 mM, whereas millimolar Mg-ATP restored the activity after run-down. The run-down channels could never be reactivated by ATP in the absence of Mg²⁺, or by a nonhydrolyzable ATP analog, AMPPNP, even in the presence of Mg²⁺.     

The Ca2+-sensitive K+-currents underlying the slow afterhyperpolarization of bullfrog sympathetic neurones

Ca²⁺-sensitive K⁺ currents involved in the slow afterhyperpolarization (a.h.p.) of an action potential of bullfrog sympathetic neurones were studied with a single-electrode voltage clamp method. The outward tail current (I_AH) generated after the end of a depolarizing command pulse (from the holding potential of –60 mV to 0 mV, 5–20 ms in duration), mimicking an action potential, was separated into at least two exponential components (I_AHf and I_AHs). They were identified as K⁺ currents, since their reversal potentials were close to the K⁺ equilibrium potential and they were sensitive to external K⁺. The time constant of I_AHf (t _f; 44 ms at –60 mV) was decreased by membrane hyperpolarization from –40 to –80 mV, while that of I_AHs (t _s; 213 ms) remained constant. Removal of external Ca²⁺ or addition of Cd²⁺ significantly decreased the I_AHs amplitude (A_s) andt _f without a change int _s and the I_AHf amplitude (A_f). On the other hand, increasing Ca²⁺ influx by applying repetitive command pulses enhanced both A_f and A_s with negligible effects ont _f andt _s, and produced a much slower component. Intracellular injection of EGTA reduced A_f with no effect ont _f, and increased A_s with a decreasedt _s. Both muscarine and (±)-tubocurarine, which reduced I_AHs, hardly affected I_AHf. These results indicate that a.h.p. is induced by the activation of two distinct Ca²⁺-dependent K⁺ channels, which differ in voltage sensitivity, Ca²⁺-dependence and pharmacology.