LVA and HVA Ca(2+) currents in ventricular muscle cells of the Lymnaea heart.

The single-electrode voltage-clamp technique was used to characterize voltage-gated Ca(2+) currents in dissociated Lymnaea heart ventricular cells. In the presence of 30 mM tetraethylammonium (TEA), two distinct Ca(2+) currents could be identified. The first current activated between -70 and -60 mV. It was fully available for activation at potentials more negative than -80 mV. The current was fast to activate and inactivate. The inactivation of the current was voltage dependent. The current was larger when it was carried by Ca(2+) compared with Ba(2+), although changing the permeant ion had no observable effect on the kinetics of the evoked currents. The current was blocked by Co(2+) and La(3+) (1 mM) but was particularly sensitive to Ni(2+) ions ( approximately 50% block with 100 microM Ni(2+)) and insensitive to low doses of the dihydropyridine Ca(2+) channel antagonist, nifedipine. All these properties classify this current as a member of the low-voltage-activated (LVA) T-type family of Ca(2+) currents. The activation threshold of the current (-70 mV) suggests that it has a role in pacemaking and action potential generation. Muscle contractions were first seen at -50 mV, indicating that this current might supply some of the Ca(2+) necessary for excitation-contraction coupling. The second, a high-voltage-activated (HVA) current, activated at potentials between -40 and -30 mV and was fully available for activation at potentials more negative than -60 mV. This current was also fast to activate and with Ca(2+) as the permeant ion, inactivated completely during the 200-ms voltage step. Substitution of Ba(2+) for Ca(2+) increased the amplitude of the current and significantly slowed the rate of inactivation. The inactivation of this current appeared to be current rather than voltage dependent. This current was blocked by Co(2+) and La(3+) ions (1 mM) but was sensitive to micromolar concentrations of nifedipine ( approximately 50% block 10 microM nifedipine) that were ineffective at blocking the LVA current. These properties characterize this current as a L-type Ca(2+) current. The voltage sensitivity of this current suggests that it is also important in generating the spontaneous action potentials, and in providing some of the Ca(2+) necessary for excitation-contraction coupling. These data provide the first detailed description of the voltage-dependent Ca(2+) currents present in the heart muscle cells of an invertebrate and indicate that pacemaking in the molluscan heart has some similarities with that of the mammalian heart.

FMRFamide-activated Ca2+ channels in Lymnaea heart cells are modulated by "SEEPLY," a neuropeptide encoded on the same gene.

The cell-attached, patch-clamp technique was used to investigate the modulatory role of the neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamide-activated Ca2+ channels in isolated Lymnaea heart ventricular cells. Both SEEPLY and FMRFamide are encoded on the same neuropeptide gene and are coexpressed in a pair of excitatory motor neurons that innervate the heart. FMRFamide applied alone was capable of significantly increasing the P(open) time of a Ca2+ channel in isolated heart muscle cells. However, SEEPLY applied alone did not significantly alter the basal level of Ca2+ channel activity in the same cells. Repeated applications of FMRFamide (15 s every min) resulted in a progressive reduction in the number of Ca2+ channel openings and the overall P(open) time of the channel. The fifth successive 15-s application of FMRFamide failed to cause the Ca2+ channels to open in the majority of cells tested. When FMRFamide and SEEPLY were repeatedly applied together (2-min applications every 4 min) the FMRFamide-activated Ca2+ channels continued to respond after the fifth application of the two peptides. Indeed channel activity was seen to continue after repeated 2-min applications of FMRFamide and SEEPLY for as long as the patch lasted (

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro.

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78: 2235-2245, 1997. The hyperpolarization-activated current (Ih) underlying inward rectification in neurons of the rat's dorsal nucleus of the lateral lemniscus (DNLL) was investigated using whole cell patch-clamp techniques. Patch recordings were made from DNLL neurons of young rats (21-30 days old) in 400 micro;m tissue slices. Under current clamp, injection of negative current produced a graded hyperpolarization of the cell membrane, often with a gradual sag in the membrane potential toward the resting value. The rate and magnitude of the sag depended on the amount of hyperpolarizing current. Larger current resulted in a larger and faster decay of the voltage. Under voltage clamp, hyperpolarizing voltage steps elicited a slowly activating inward current that was presumably responsible for the sag observed in the voltage response to a steady hyperpolarizing current recorded under current clamp. Activation of the inward current (Ih) was voltage and time dependent. The current just was seen at a membrane potential of -70 mV and was activated fully at -140 mV. The voltage value of half-maximal activation of Ih was -78.0 +/- 6.0 (SE) mV. The rate of Ih activation was best approximated by a single exponential function with a time constant that was voltage dependent, ranging from 276 +/- 27 ms at -100 mV to 186 +/- 11 ms at -140 mV. Reversal potential (Eh) of Ih current was more positive than the resting potential. Raising the extracellular potassium concentration shifted Eh to a more depolarized value, whereas lowering the extracellular sodium concentration shifted Eh in a more negative direction. Ih was sensitive to extracellular cesium but relatively insensitive to extracellular barium. The current amplitude near maximal-activation (about -140 mV) was reduced to 40% of control by 1 mM cesium but was reduced to only 71% of control by 2 mM barium. When the membrane potential was near the resting potential (about -60 mV), cesium had no effect on the membrane potential, current-evoked firing rate and input resistance but reduced the spontaneous firing. When the membrane potential was more negative than -70 mV, cesium hyperpolarized the cell, decreased current-evoked firing and increased the input resistance. Ih in DNLL neurons does not contribute to the normal resting potential but may enhance the extent of excitation, thereby making the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.

Synaptic excitation in the dorsal nucleus of the lateral lemniscus: whole-cell patch-clamp recordings from rat brain slice.

The synaptic events underlying the excitation of neurons in the rat's dorsal nucleus of the lateral lemniscus were studied by whole-cell patch-clamp recordings in a brain slice preparation of the auditory midbrain. Both current-clamp and voltage-clamp data were obtained with the brain slice submerged in artificial cerebrospinal fluid. The rats were between 21 and 35 days of age at the time the recordings were made. Synaptic responses were evoked by a bipolar stimulating electrode placed on the lateral lemniscus just ventral to the dorsal nucleus. To eliminate glycinergic inhibitory responses, all physiological data were gathered with 0.5 microM strychnine added to the saline bath. Under current-clamp conditions, excitatory postsynaptic potentials could be subdivided into early and late components. The early component produced a single, highly reliable, short-latency spike and the later component produced a more variable, long-latency spike or train of spikes. The non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, completely blocked the early excitatory postsynaptic potential and its associated action potential. The N-methyl-D-aspartate antagonist, D,L-2-amino-5-phosphonovaleric acid, blocked the later excitatory postsynaptic potential and its action potentials. Typically, both early and late excitatory postsynaptic potentials could be recorded from the same cell, but the early excitatory postsynaptic potential was evoked at lower stimulus levels and had a larger amplitude than the later excitatory postsynaptic potential. Under voltage-clamp conditions, dorsal nucleus of the lateral lemniscus neurons responded to stimulation of the lateral lemniscus with excitatory postsynaptic currents. Outward excitatory postsynaptic currents were recorded with holding potentials that depolarized the cell membrane and inward currents were seen when the cell was hyperpolarized. The current-voltage (I-V) relation of the early peak portion of the excitatory postsynaptic current was nearly linear, whereas the I-V relation of the later excitatory postsynaptic current (12 ms after the peak) was non-linear over the range between -50 and - 100 mV. The outward excitatory postsynaptic current consisted of an early current that was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and a later current that was blocked by D,L-2-amino-5-phosphonovaleric acid. In artificial cerebrospinal fluid with normal concentrations of Mg2+, the inward excitatory postsynaptic current was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, but was not affected by D,L-2-amino-5-phosphonovaleric acid. In Mg2+-free artificial cerebrospinal fluid. however, the early component of the inward excitatory postsynaptic current was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and a later component was blocked by D,L-2-amino-5-phosphonovaleric acid. The results indicate that both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated synaptic responses are present in dorsal nucleus of the lateral lemniscus neurons of rats at 21-35 days of age. The N-methyl-D-aspartate component had a longer time-course and a higher threshold than the non-N-methyl-D-aspartate component, and was subject to a voltage-dependent Mg2+ block when the cell's membrane was hyperpolarized. The long-duration N-methyl-D-aspartate component is probably responsible for the prolonged inhibitory effect of dorsal nucleus of the lateral lemniscus neurons on physiological responses in the rat's inferior colliculus.

Potassium currents and membrane excitability of neurons in the rat's dorsal nucleus of the lateral lemniscus.

1. The contribution of voltage-activated outward potassium currents to membrane excitability of neurons in the rat's dorsal nucleus of the lateral lemniscus (DNLL) was studied in a brain slice preparation using whole cell patch-clamp and intracellular recordings. Voltage-clamp methods and pharmacological manipulations were used to examine the currents regulating membrane dynamics in DNLL. 2. A delayed sustained outward current was evoked by applying depolarizing voltage steps across the cell membrane from a holding potential of -50 mV. An additional transient outward current was evoked when the depolarizing steps were preceded by a hyperpolarizing prepulse of -110 or -120 mV. 3. The transient outward current peaked within 6.8 ms of the onset of a depolarizing pulse. It decayed with a time constant of 12.3 ms for a 60-mV depolarizing voltage shift. Half-inactivation of this current occurred at -81.3 mV. The time constant for removal of the inactivation was 17.4 ms. The transient current had a high sensitivity to 4-aminopyridine (4-AP). 4. The sustained current was activated more slowly and was more sensitive to tetraethylammonium (TEA) than the transient current. The sustained current had both Ca2+-dependent and Ca2+-independent components. The Ca2+-dependent portion emerged at potentials of about -35 mV and was activated fully at +10 mV. The Ca2+-independent component was activated at potentials more positive than -40 mV and increased in magnitude with further depolarization. Inactivation of the Ca2+-independent component was voltage dependent. Also, TEA suppressed the Ca2+-independent compound. 5. The transient current in DNLL neurons closely resembled the A current (IA) described for hippocampal and other neurons in both kinetics and pharmacology. The Ca2+-independent component of the sustained current resembled the K current (IK) described for other neurons in both its properties of activation and inactivation and its pharmacology. 6. The outward current of some DNLL neurons was found to contain a dendrotoxin-sensitive component. This component reached its peak at 6.8 ms and had voltage-sensitive time constants of decay of 25.5 and 8.5 ms with voltage steps of 40 and 60 mV, respectively. 7. Application of 4-AP and TEA markedly prolonged the spike width, abolished the fast component of the after hyperpolarization and depolarized the cell membrane. Also, the number of action potentials produced by positive current injection increased under the influence of 4-AP and TEA. Membrane excitability and spike repolarization were dependent on both 4-AP-sensitive transient and TEA-sensitive sustained currents. 8. Neurons in DNLL typically exhibit a steady discharge of action potentials in response to sustained membrane depolarization. The rate and temporal pattern of production of action potentials in these cells are determined by the combination of transient and sustained potassium channels.

A review of the electrophysiological, pharmacological and single channel properties of heart ventricle muscle cells in the snail Lymnaea stagnalis.

Although a considerable body of information has accumulated describing the pharmacological properties of a wide range of molluscan muscle types, the physiological bases underlying these properties have not been thoroughly investigated. At present, little is known about the types of ion channels and their regulation in molluscan muscle cell membranes. Voltage-clamp, and more recently, patch-clamp techniques have revealed molluscan muscles possess a complex array of channel types with various pharmacological and electrophysiological properties. The gating properties of these channels and their modulation by chemical agents, however, are still poorly understood. This review summarizes some aspects of molluscan muscle function with particular reference to the heart ventricle muscle of the pond snail, Lymnaea stagnalis.

Activation of a divalent cation-conducting channel in heart ventricle muscle cells of the snail Lymnaea stagnalis by the molluscan cardioactive peptide FMRFamide.

Ion channels with characteristics of Ca2+ channels have been found in isolated heart ventricle cells of the snail Lymnaea stagnalis. Although spontaneous Ca2+ or Ba2+ currents were seen only occasionally, spontaneous inward Na+ currents were readily observed in the absence of patch pipette Ca2+ between membrane potentials of -100 mV and +20 mV. These currents were blocked by 2 mM Ni2+, 2 mM Co2+ and 10 microM Ca2+. The channels usually ceased conducting within a few minutes after seal formation with the patch pipette and could not be re-activated with depolarizing voltage steps. However, at the cell's resting potential, 10(-8) to 10(-6) M of the molluscan cardioactive peptide FMRFamide or its analogue FLRFamide2+ applied to the cell membrane away from the patch pipette, induced unitary Ba2+ currents or, in the absence of Ca2+ in the patch pipette, Na+ currents. This suggests that a secondary messenger is involved in the FMRFamide-induced activation of these channels rather than a direct activation of a channel-receptor complex by the peptide.

The peptide FMRFamide activates a divalent cation-conducting channel in heart muscle cells of the snail Lymnaea stagnalis.

1. Isolated Lymnaea stagnalis heart ventricle cells contain cation-conducting channels with properties characteristic of Ca2+ channels. These channels, which carry inward Na+ currents in the absence of Ca2+, are activated by the molluscan cardioactive peptides FMRFamide and FLRFamide, and are blocked by Co2+ ions. 2. FMRFamide also activated inward Ba2+ currents at the cell's resting potential. These currents, which were also blocked by Co2+ ions, reversed at a membrane potential of +70 mV. 3. Both sodium and barium currents were initiated when the peptides were applied to the cell outside of the patch pipette indicating that a secondary messenger is likely to be involved in the FMRFamide response.

Non-voltage-gated calcium channels in snail heart ventricle cells.

1. Two recently identified channel types in Lymnaea stagnalis heart muscle cells were shown to conduct Na+ in the absence of extracellular Ca2+. They did not appear to be 'voltage-gated' as they were not activated by voltage. Also, they remained active over a wide range of membrane potentials. However, they were weakly 'voltage-sensitive' as their activity usually tended to increase with depolarization. The weak voltage-sensitivity and similarity to other non-voltagegated Ca2+ channels suggested that one or both of these channels may be receptor-operated Ca2+ channels. 2. One of the two channels had a slope conductance of 15 pS. The other appeared to have at least two subconductance states with slope conductances of 50 and 72pS. Both these conductance states had very similar open dwell-time constants and identical reversal potentials. The open dwell-time constants of both conductance states were not affected by voltage, suggesting that the channels' weak voltage-sensitivity was mediated by one of the closed states. 3. With divalent cations in the patch pipette, non-voltage-gated Ba2+ and Ca2+ currents were also detected. The Ba2+ conductance (12pS) was similar to the Ca2+ conductance (11pS).

The sites of action of praziquantel in a smooth muscle of Lymnaea stagnalis.

Praziquantel (1.6 X 10(-6) M), a new anthelmintic drug, was shown to produce a sustained contracture in the penis retractor muscle of the freshwater snail Lymnaea stagnalis. Calcium-free saline greatly reduced the magnitude and duration of the praziquantel contractures if the preparation had been previously exposed to praziquantel. The praziquantel contracture in zero-calcium saline was abolished if the preparation was pretreated with successive applications of 2 X 10(-2) M caffeine. Lanthanum (1.0 mM) in zero-calcium saline prevented the contractures normally produced by 30 mM potassium saline, but did not prevent the praziquantel contracture. In normal saline, however, 1.0 mM lanthanum greatly reduced the magnitude of a praziquantel-induced contracture. We conclude that praziquantel increases sarcolemmal calcium permeability and releases calcium from intracellular stores in Lymnaea stagnalis smooth muscle.