Ca-dependent Current Research Articles

BK Channel Regulation of Afterpotentials and Burst Firing in Cerebellar Purkinje Neurons.

BK calcium-activated potassium channels have complex kinetics because they are activated by both voltage and cytoplasmic calcium. The timing of BK activation and deactivation during action potentials determines their functional role in regulating firing patterns but is difficult to predict a priori. We used action potential clamp to characterize the kinetics of voltage-dependent calcium current and BK current during action potentials in Purkinje neurons from mice of both sexes, using acutely dissociated neurons that enabled rapid voltage clamp at 37°C. With both depolarizing voltage steps and action potential waveforms, BK current was entirely dependent on calcium entry through voltage-dependent calcium channels. With voltage steps, BK current greatly outweighed the triggering calcium current, with only a brief, small net inward calcium current before Ca-activated BK current dominated the total Ca-dependent current. During action potential waveforms, although BK current activated with only a short (∼100 μs) delay after calcium current, the two currents were largely separated, with calcium current flowing during the falling phase of the action potential and most BK current flowing over several milliseconds after repolarization. Step depolarizations activated both an iberiotoxin-sensitive BK component with rapid activation and deactivation kinetics and a slower-gating iberiotoxin-resistant component. During action potential firing, however, almost all BK current came from the faster-gating iberiotoxin-sensitive channels, even during bursts of action potentials. Inhibiting BK current had little effect on action potential width or a fast afterhyperpolarization but converted a medium afterhyperpolarization to an afterdepolarization and could convert tonic firing of single action potentials to burst firing.SIGNIFICANCE STATEMENT BK calcium-activated potassium channels are widely expressed in central neurons. Altered function of BK channels is associated with epilepsy and other neuronal disorders, including cerebellar ataxia. The functional role of BK in regulating neuronal firing patterns is highly dependent on the context of other channels and varies widely among different types of neurons. Most commonly, BK channels are activated during action potentials and help produce a fast afterhyperpolarization. We find that in Purkinje neurons BK current flows primarily after the fast afterhyperpolarization and helps to prevent a later afterdepolarization from producing rapid burst firing, enabling typical regular tonic firing.

Contribution of Ca-Regulated Ion Currents to the Action Potential Morphology During Cardiac Alternans

Cardiac alternans, described as periodic beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is recognized as high risk indicator for cardiac arrhythmias, stroke and sudden cardiac death.We investigated mechanisms of cardiac alternans in single rabbit atrial myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique, and revealed a strong correlation between CaT amplitude and AP duration (APD) during alternans. Investigation of [Ca]i-Vm interactions indicates that disturbances in Ca signaling are the primary events mediating activity of Ca-regulated ion channels and leading to the changes in AP morphology. Voltage-clamp in form of pre-recorded APs (AP clamp) was used to assess the contribution of Ca-regulated membrane currents by measuring the difference between currents recorded during large and small alternans CaTs. Ca-dependent currents exhibited a large outward component (2.9 ± 1.1 pA/pF in amplitude) during AP phases 1 and 2 that was followed by an inward current (0.5 ± 0.1 pA/pF in amplitude; n=7) during AP repolarization. We also investigated contributions of Na/Ca exchange (NCX), L-type Ca, Ca-activated Cl and small conductance Ca-activated K currents to the AP morphology during Ca alternans. ∼90 % of the initial outward current was blocked by substitution of Cl ions or application of Cl channel blocker (DIDS) indicating that this current is mainly carried by Ca-activated Cl channels. In summary, during Ca alternans the prominent prolongation of AP at APD10 to APD30 repolarization levels during the small CaT is due to reduced Ca-activated Cl current while the overall effect of other Ca-sensitive currents (NCX, L-type Ca, small conductance K) cause slight shortening of AP in the APD50 to APD90 range.

Dynamic Changes Of Local Ca Sensed By Ca-dependent Currents In Cardiac Myocytes

In cardiac cells, Ca release from the sarcoplasmic reticulum (SR) is a local event that occurs in the subsarcolemmal space. In this study, we investigated dynamic changes of subsarcolemmal Ca sensed by Ca current (ICa) and Na/Ca exchanger (NCX) during SR Ca release.In pig ventricular myocytes, membrane currents were recorded using whole-cell voltage-clamp with Fluo-3 as indicator for global Ca. SR Ca release was triggered through activation of ICa during steps from -70 to -35 mV. At this potential, ICa showed release-dependent inactivation and recovery. The step at -35 mV was interrupted at different time intervals by a step to -70 mV or 0 mV to measure the time course of NCX and availability of ICa respectively. NCX tail currents were converted to subsarcolemmal Ca using steady-state dependence of NCX on global Ca during caffeine application. Release-dependent inactivation of ICa at +10 mV was assessed by subtraction analysis of two pulses with different amplitudes of Ca release during repetitive stimulation in Na-free conditions after caffeine.Subsarcolemmal Ca reached its peak value immediately after the trigger pulse, where global Ca increased more slowly and to a lesser extent. Maximal inactivation and recovery of ICa occurred 20-30 ms after the step to -35 mV, with a faster time course than changes in global Ca, but slower than maximal NCX activation. At a more positive potential of +10 mV, inactivation of ICa was maximal at 12.7±0.78 ms (n=10).In conclusion, local Ca sensed by NCX and ICa during triggered release considerably differs from changes in global Ca. The discrepancy between time courses of local Ca effects on NCX and ICa is currently unexplained and may be related to the longer latency for Ca channels at more negative potentials.

Ca(2+)-dependent Cl- conductance in taste cells from Necturus.

1. Taste responses adapt to a constant chemical stimulus. The present study describes a new ionic conductance in taste cells--a Ca(2+)-dependent anion conductance that may explain taste adaptation. 2. Patch-clamp recordings were made on isolated Necturus taste cells or on taste cells in lingual slices. When Na+ and K+ currents were eliminated with tetrodotoxin (TTX) and tetraethyl-ammonium (TEA) in the bath and replacing K+ with N-methyl-D-glucamine (NMDG+) in the pipette, Ca2+ currents were followed by prolonged outward currents. Outward current was abolished when Ca2+ was substituted with Ba2+ or when Cl- was replaced with large organic anions (methanesulfonate, isethionate, or ascorbate). 3. The outward, Ca-dependent current was reduced by certain agents that block Cl- conductances in other tissues, namely 4-acet-amido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). However, other Cl- channel blockers--9-AC, furosemide and an antibody to Cl channels-had little or no specific effect on the Ca-dependent outward current in Necturus taste cells. 4. We postulate that the biological action of this Ca-dependent anion conductance in situ is to terminate depolarizing receptor potentials, even during maintained chemostimulation, thereby playing an important role in chemosensory adaptation and modulation of impulse discharge patterns in taste buds.

Characteristic firing behavior of cell types in the cardiorespiratory region of the nucleus tractus solitarii of the rat

The present in vitro study was performed to characterize neurons within dorsal regions of the nucleus tractus solitarii (NTS), principally at the level of area postrema, and known to recieve inputs predominantly from cardiovascular and respiratory afferents (i.e. cardiorespiratory NTS). This report describes 4 classes of neurons (S1–S4) that were silent at their resting membrane potential and received relatively short (<3.6 ms). and consistent latency synaptic inputs (±0.4 ms) comprising either an EPSP or EPSP/IPSP sequence following low intensity electrical stimulation of the solitary tract (ts). Intracellular recording with sharp electrodes were used to characterize neuron types based on their different firing response patterns to injection of depolarizing current. S1 cells showed a single action potential; s2 fired repetitively; S3 produced a 2–5 spike burst coincident with the start of the current pulse and S4 neurons showed delayed exicitation. Accommodation of firing frequency was seen in S2, S3 and some S4 cells. The voltage dependency of the different discharge patterns of the 4 cell groups was tested by current pulse stimulation at different holding potentials. However, in the majority of cells in any one cell class the firing pattern was qualitatively similar. Based on these findings it is suggested that the different firing characteristics in intrinsic membrane properties between neuron classes. Representative examples from each of the defined cell classes were further studied in current and voltage clamp using the whole cell patch technique to define the presence and role of certain ionic currents in the firing response patterns of the cell groups. In the current clamp configuration the firing behavior of S1 neurons (single spiking) was unaltered during exposure to 4-aminopyridine (4-AP; 2 mM), cobalt of this neuron is due (Co; 5 mM), norepinephrine (NE; 20 μM) and muscarine chloride (50 μM). It is suggested that the relatively low excitability of this neuron is due a persistent outward current which occurred at −40 mV during depolarizing voltage steps in the voltage clamp configuration. A common characteristic of S2 neurons (repetitively firing) was that they showed accommodation during current injection which was greatly attenuated in the presence of Co or NE. In addition, 4-AP slowed the firing frequency, reduced the afterhyperpolarization and broadened the spike width of S2 cells. Interestingly, the amount of accommodation observed in S2 cells was variable for cells of this class and was proportional to the magnitude of a Co-sensitive inward current present during depolarizing voltage steps between −45 to −5 mV. Exposure of S3 cells to NE resulted in repetitive firing with reduced accommodation during current pulse injection. The frequency of discharge during a current pulse was further increased by subsequent administration of 4-AP. Finally, the magnitude of the delayed excitation of S4 neurons was dependent on the voltage and was abolished by 4-AP. This was consistent with voltage clamp data showing a voltage-dependent and 4-AP sensitive outward current, first evident at −40 mV. The present study demonstrates the presence of 4 different cell types within the dorsal or cardiorespiratory region of the NTS. The distinction between the 4 cell groups are based on firing response pattern to injected current and these differences correlate with qualitative differences in the magnitude, time course and voltage dependency of : (1) a 4-AP sensitive current, (2) a Ca, and (3) Ca-dependent currents sensitive to NE and apamin. It is suggested that the intrinsic membrane properties of neurons within the cardiorespiratory NTS may play a role in the integration of afferent inputs. In order to explore these possibilities the following paper describes computational model neurons based on the present data and a previous study on rhythmically active neurons (Paton et al., J. Neurophysiol., 66 (1991) 824–838).

Ionic currents in the soma of an identified cockroach motoneurone recorded under voltage-clamp

1. 1. Membrane currents have been recorded from the soma of a bifunctional basalar/coxal depressor motoneurone in the metathoracic ganglion of the cockroach ( Periplaneta americana) using a two-electrode voltage-clamp technique. 2. 2. This motoneurone cell body is normally inexcitable when studied under current-clamp. Appropriate depolarizing command steps evoke rapid transient outward currents and late outward currents. 3. 3. Late outwart currents are dominated by a Ca-dependent component that confers and N-shaped I-V relationship on the neurone. 4. 4. The Ca-dependent outward current is suppressed by Cd 2+ (1 mM), Mn 2+ (5mM) or verapamil (50 μM). 5. 5. Externally applied tetraethylammonium ions (TEA + (25 mM) block the Ca-dependent current, but also appear to suppress a component of the late outward current that is independent of Ca 2+. also appears to suppress a component of the late outward current that is independent of Ca 2+. 6. 6. Aminopyridines cause only minor suppression of late outward currents, but shift the peak in the N-shaped I- V relationship to more negative potentials. 7. 7. The reversal potential of tail currents recorded following pre-pulses to + 50 mV were dependent upon the pre-pulse duration; increasing the duration from 10 to 50 msec caused a + 17 mV shift in tail current reversal potential. 8. 8. A five-fold increase in the K + concentration of the solution bathing the preparation only produced small and incosistent changes in the reversal potential of tail currents. 9. 9. Five-fold reduction in external Cl − caused no change. 10. 10. The dependence of tail current reversal potential upon pre-pulse duration and the limited effect of alterations in the composition of the bathing solution are discussed in the context of restricted ion movements near the external surface of the cell membrane.

Dependence of intracellular effects of GTP gamma S and inositoltrisphosphate on cell membrane potential and on external Ca ions.

Single cells from rat lacrimal glands were studied with the tight-seal whole-cell recording method. Ca-dependent K and Cl currents were measured in response to dialysis with inositoltrisphosphate or GTP gamma S, two compounds known for elevating internal Ca2+ concentration. The activation of the Ca-dependent currents elicited by either compound was partially inhibited by sustained depolarization or by removal of external Ca2+. Conversely, hyperpolarization or removal of external Mg2+ led to augmentations of the Ca-dependent currents. These effects became apparent about 1 min after initiation of cell dialysis with inositoltrisphosphate- or GTP gamma S-containing solutions, and they further developed during the ensuing 10 min. Holding potential and external divalent cations did not affect the Ca-dependent currents elicited by dialysing the cells with strongly buffered solutions containing 0.5 microM free Ca2+. In Ca2+-free external saline, cell currents were independent of the holding potential. It is suggested that InsP3 augments intracellular Ca2+ levels not only by releasing Ca2+ from internal stores but also by slowly increasing the Ca permeability of the plasma membrane. The results indicate that Ca2+ entry through the plasma membrane increases with hyperpolarization. The similarity of the effects seen in InsP3- and GTP gamma S-dialysed cells gives support to the hypothesis that production of the former depends on a GTP-binding protein.

Acetylcholine modulation of the conductance of intercellular junctions between rat lacrimal cells.

The conductance of intercellular junctions between rat lacrimal cells was studied with the double whole-cell tight-seal recording technique. This conductance decreases spontaneously with time as a result of the double-cell dialysis. The rate of this 'spontaneous' uncoupling is unaffected by changing the internal Ca concentration, [Ca]i, between 10(-8) M and 10(-6) M. This rate of uncoupling is greatly increased when [Ca]i is approximately 10(-5) M, and this effect does not involve changes in the internal proton concentration. When [Ca]i is weakly buffered in one of the two cells, 1-2 microM-acetylcholine (ACh) both activates Ca-dependent channels in that cell (Marty, Tan & Trautmann, 1984) and uncouples the two cells. The uncoupling is not synchronous with the increase in [Ca]i as reflected by the Ca-dependent currents. When [Ca]i is strongly buffered in both cells, ACh fails to activate Ca-dependent currents, but it can still uncouple the cells. This ACh-induced uncoupling is often preceded by a transient enhancing of the coupling. In conclusion, ACh has several distinct effects on lacrimal cells: activation of Ca-dependent channels in the plasma membrane, closure of junctional channels involving a Ca-independent mechanism, and sometimes, an increase in the junctional coupling by a Ca-independent mechanism.

Potassium channels in cultured bovine adrenal chromaffin cells.

K channels of bovine adrenal chromaffin cells were studied using patch-clamp techniques. Whole-cell K currents measured near +10 mV were much larger in 1 mM-external Ca than in Ca-free saline. Noise analysis suggested that this Ca-dependent current was carried by a large unitary conductance channel, called BK channel, which was previously described in inside-out patches (Marty, 1981). The Ca-dependent K current near +10 mV declined with time due to 'run-down' of Ca channels. At the same time, a fraction of the outward current observed above +50 mV was also eliminated. This outward current component probably represents K efflux through Ca channels. Whole-cell Ca-dependent K currents were studied using various Ca buffers. EGTA buffers were surprisingly inefficient: in order to block the current entirely, it was necessary to use an isotonic EGTA solution and to increase internal pH. 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was at least five times more efficient than EGTA. In isolated patches three types of single-channel K currents were observed. Under normal ionic conditions (140 mM-K inside, 140 mM-Na outside), the unitary conductances measured between -20 and +40 mV were 96 pS, 18 pS and 8 pS. The 96 pS channels are the Ca-dependent BK channels. 18 pS and 8 pS channels were both activated and then inactivated by membrane depolarization. Both displayed complex kinetics; single-channel currents were grouped in bursts. Activation and inactivation kinetics were faster for the 18 pS channel (therefore termed FK channel, for fast K channel) than for the 8 pS channel (SK channel, for slow or small amplitude channel). The voltage dependence of opening probability was steeper for the FK channel as compared to the SK channel.

Two components of Ca-dependent potassium current in identified neurones ofAplysia californica

Outward tail currents measured in Aplysia neurones after termination of depolarizing voltage-clamp pulses consist of rapidly decaying voltage-dependent K currents and slow tail currents of much slower time course. The rapidly decaying voltage-dependent tail currents were blocked with aminopyridines, and measurements of the slow tail currents were made following decay of any residual rapid tail currents. The slow tail current exhibited two components of differing sensitivity to externally applied tetraethylammonium (TEA) ions. In some neurones of the abdominal ganglion (L-2, L-4), virtually all of the slow tail current was resistant to blockage by TEA, while in others (L-3, L-6) 80% or more of the slow tail current was blocked by low TEA concentrations (KD less than 1 mM), the remaining slow tail current being resistant to TEA. This TEA-resistant slow tail current was identified as a K current because it reversed near the K equilibrium potential (EK), the reversal potential was shifted by changes in the external K concentration, and it could be blocked by injection of Cs+. It was abolished by replacement of external Ca2+ by Co2+ or Ba2+, by addition of Cd2+, or by injection of EGTA, and thus determined to be a Ca-dependent current. Intracellular injection of TEA or external application of aminopyridine or apamine had little or no effect on the TEA-resistant slow tail current. Quinidine reduced the TEA-sensitive, but not the TEA-resistant current. Both the TEA-sensitive and the TEA-resistant components of the slow tail current exhibited similar time courses of decay.(ABSTRACT TRUNCATED AT 250 WORDS)

Cadmium-induced blockade of the cardiac fast Na channels in calf Purkinje fibres.

The fast transient inward current elicited by depolarizations above about -60 mV in calf Purkinje fibres was found to be depressed by Cd in concentrations less than 1 mM. The Cd-sensitive current, which strongly depended on external Na, was recorded in the presence of 2 mM MnCl2 and was blocked by TTX, indicating that a contamination from slow Ca-dependent currents could be discounted. The current reduction caused by Cd was also observed in nominally Ca-free solutions. The Cd-induced depression of the fast Na current was not accompanied by changes in the current kinetic parameters, as revealed by comparing inactivation curves and peak current voltage relations at different Cd concentrations, and could be attributed to a voltage-independent channel blocking action. Half-blockade occurred at 0.182 +/- 0.06 mM (n = 4). Plots of peak current amplitude as a function of the Cd concentration showed that the cooperation of two Cd ions was required to block a single channel.