Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma.
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Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma. The cell body of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) displays a time-dependent change in excitability. Immediately after dissection, depolarization evokes plateau potentials, but after several hours all-or-none action potentials are evoked. Because K channel blockers have been shown to produce a similar transition in electrical properties, we have used current-clamp, voltage-clamp and action-potential-clamp recording to elucidate the contribution of different classes of K channel to the transition in electrical activity of the neuron. Apamin had no detectable effect on the neuron, but charybdotoxin (ChTX) caused a rapid transition from plateau potentials to spikes in the somatic response of Df to depolarization. In neurons that already produced spikes when depolarized, ChTX increased spike amplitude but did not increase their duration nor decrease the amplitude of their afterhyperpolarization. 4-Aminopyridine (4-AP) (which selectively blocks transient K currents) did not cause a transition from plateau potentials to spikes but did enhance oscillations superimposed on plateau potentials. When applied to neurons that already generated spikes when depolarized, 4-AP could augment spike amplitude, decrease the latency to the first spike, and prolong the afterhyperpolarization. Evidence suggests that the time-dependent transition in electrical properties of this motoneuron soma may result, at least in part, from a fall in calcium-dependent potassium current (IK,Ca), consequent on a gradual reduction in [Ca2+ ]i. Voltage-clamp experiments demonstrated directly that outward K currents in this neuron do fall with a time course that could be significant in the transition of electrical properties. Voltage-clamp experiments also confirmed the ineffectiveness of apamin and showed that ChTX blocked most of IK,Ca. Application of Cd2+ (0.5 mM), however, caused a small additional suppression in outward current. Calcium-insensitive outward currents could be divided into transient (4-AP-sensitive) and sustained components. The action-potential-clamp technique revealed that the ChTX-sensitive current underwent sufficient activation during the depolarizing phase of plateau potentials to enable it to shunt inward conductances. Although the ChTX-sensitive conductance apparently makes little contribution to spike repolarization, the ChTX-resistant IK,Ca does make a significant contribution to this phase of the action potential. The 4-AP-sensitive current began to develop during the rising phase of both action potentials and plateau potentials but had little effect on the electrical activity of the neuron, probably because of its relatively small amplitude. (+info)
Acute repair of crushed guinea pig spinal cord by polyethylene glycol.
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Acute repair of crushed guinea pig spinal cord by polyethylene glycol. We have studied the responses of adult guinea pig spinal cord white matter to a standardized compression within a sucrose gap recording chamber. This injury eliminated compound action potential (CAP) conduction through the lesion, followed by little or no recovery of conduction by 1 h postinjury. We tested the ability of polyethylene glycol (PEG) to repair the injured axons and restore physiological function. Local application of PEG (1,800 MW, 50% by weight in water) for approximately 2 min restored CAP conduction through the injury as early as 1 min post PEG application. The recovery of the CAP +info)
Receptor potentials and electrical properties of nonspiking stretch-receptive neurons in the sand crab Emerita analoga (Anomura, Hippidae).
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Receptor potentials and electrical properties of nonspiking stretch-receptive neurons in the sand crab Emerita analoga (Anomura, Hippidae). Four nonspiking, monopolar neurons with central somata and large peripheral dendrites constitute the sole innervation of the telson-uropod elastic strand stretch receptor in Emerita analoga. We characterized their responses to stretch and current injection, using two-electrode current clamp, in intact cells and in two types of isolated peripheral dendritic segments, one that included and one that excluded the dendritic termini (mechanosensory membrane). The membrane potentials of intact cells at rest (mean +/- SD: -57 +/- 4. 4 mV, n = 30), recorded in peripheral or neuropil processes, are similar to the membrane potentials of isolated dendritic segments and always less negative than membrane potentials of motoneurons and interneurons recorded in the same preparations. Ion substitution experiments indicate that the membrane potential is influenced strongly by Na+ conductance, probably localized in the mechanotransducing terminals within the elastic strand. The form of the receptor potential in response to ramp-hold-release stretch remains the same as stretch amplitude is varied and is not dependent on initial membrane potential (-70 to -30 mV) or recording site: initial depolarization (slope follows ramp of applied stretch), terminated by rapid, partial repolarization to a plateau (delayed depolarization) that is intermediate between the peak depolarization and the initial potential and sustained for the duration of the stretch. Responses to depolarizing current pulses are similar to stretch-evoked receptor potentials, except for small amplitude stimuli: an initial peak occurs only in response to stretch and probably reflects elastic recoil of the extracellular matrix surrounding the dendritic terminals. The rapid, partial repolarization depends on holding potential and is abolished by 4-aminopyridine (4-AP; 10 mM), implicating a fast-activating, fast-inactivating K+ conductance; TEA (60 mM) abolishes the remaining slow repolarization to the plateau. In intact cells, but not dendritic segments, regenerative depolarizations can arise in response to stretch or depolarizing current pulses; they are reduced by CdCl2 (10 microM) in the saline containing TEA and 4-AP and probably reflect current spread from Ca2+ influx at presynaptic terminals in the ganglion. We found no evidence for other voltage-activated conductances. Unlike morphologically similar "nonspiking" thoracic receptors of other species, E. analoga's nonspiking neurons are electrically compact and do not boost the analogue afferent signal by voltage-activated inward currents. The most prominent (only?) voltage-activated extra-ganglionic conductances are for potassium; by reducing the slope of the stretch-plateau depolarization curve, they extend each neuron's functional range to the full range of sensitivity of the receptor. (+info)
Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation.
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Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation. The effects of combined O2/glucose deprivation were investigated on large aspiny (LA) interneurons recorded from a striatal slice preparation by means of simultaneous electrophysiological and optical recordings. LA interneurons were visually identified and impaled with sharp microelectrodes loaded with the calcium (Ca2+)-sensitive dye bis-fura-2. These cells showed the morphological, electrophysiological, and pharmacological features of large striatal cholinergic interneurons. O2/glucose deprivation induced a membrane hyperpolarization coupled to a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, this [Ca2+]i elevation was more pronounced in dendritic branches rather than in the somatic region. The O2/glucose-deprivation-induced membrane hyperpolarization reversed its polarity at the potassium (K+) equilibrium potential. Both membrane hyperpolarization and [Ca2+]i rise were unaffected by TTX or by a combination of ionotropic glutamate receptors antagonists, D-2-amino-5-phosphonovaleric acid and 6cyano-7-nitroquinoxaline-2, 3-dione. Sulfonylurea glibenclamide, a blocker of ATP-sensitive K+ channels, markedly reduced the O2/glucose-deprivation-induced membrane hyperpolarization but failed to prevent the rise in [Ca2+]i. Likewise, charybdotoxin, a large K+-channel (BK) inhibitor, abolished the membrane hyperpolarization but did not produce detectable changes of [Ca2+]i elevation. A combination of high-voltage-activated Ca2+ channel blockers significantly reduced both the membrane hyperpolarization and the rise in [Ca2+]i. In a set of experiments performed without dye in the recording electrode, either intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid or external barium abolished the membrane hyperpolarization induced by O2/glucose deprivation. The hyperpolarizing effect on membrane potential was mimicked by oxotremorine, an M2-like muscarinic receptor agonist, and by baclofen, a GABAB receptor agonist. However, this membrane hyperpolarization was not coupled to an increase but rather to a decrease of the basal [Ca2+]i. Furthermore glibenclamide did not reduce the oxotremorine- and baclofen-induced membrane hyperpolarization. In conclusion, the present results suggest that in striatal LA cells, O2/glucose deprivation activates a membrane hyperpolarization that does not involve ligand-gated K+ conductances but is sensitive to barium, glibenclamide, and charybdotoxin. The increase in [Ca2+]i is partially due to influx through voltage-gated high-voltage-activated Ca2+ channels. (+info)
Mechanism of block and identification of the verapamil binding domain to HERG potassium channels.
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Calcium channel antagonists have diverse effects on cardiac electrophysiology. We studied the effects of verapamil, diltiazem, and nifedipine on HERG K+ channels that encode IKr in native heart cells. In our experiments, verapamil caused high-affinity block of HERG current (IC50=143.0 nmol/L), a value close to those reported for verapamil block of L-type Ca2+ channels, whereas diltiazem weakly blocked HERG current (IC50=17.3 micromol/L), and nifedipine did not block HERG current. Verapamil block of HERG channels was use and frequency dependent, and verapamil unbound from HERG channels at voltages near the normal cardiac cell resting potential or with drug washout. Block of HERG current by verapamil was reduced by lowering pHO, which decreases the proportion of drug in the membrane-permeable neutral form. N-methyl-verapamil, a membrane-impermeable, permanently charged verapamil analogue, blocked HERG channels only when applied intracellularly. Verapamil antagonized dofetilide block of HERG channels, which suggests that they may share a common binding site. The C-type inactivation-deficient mutations, Ser620Thr and Ser631Ala, reduced verapamil block, which is consistent with a role for C-type inactivation in high-affinity drug block, although the Ser620Thr mutation decreased verapamil block 20-fold more than the Ser631Ala mutation. Our findings suggest that verapamil enters the cell membrane in the neutral form to act at a site within the pore accessible from the intracellular side of the cell membrane, possibly involving the serine at position 620. Thus, verapamil shares high-affinity HERG channel blocking properties with other class III antiarrhythmic drugs, and this may contribute to its antiarrhythmic mechanism. (+info)
Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current.
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OBJECTIVE: The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (Ito). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells. The purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of Ito recorded from rat ventricular cells. METHODS: The cDNA encoding the rat Kv4.3 potassium channel was cloned into the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte Ito current. RESULTS: The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current. The current activated for potentials positive to -40 mV and the steady-state inactivation curve had a midpoint of -47.4 +/- 0.3 mV and a slope of 5.9 +/- 0.2 mV. Rat ventricular Ito current was activated at potentials positive to -20 mV and inactivated with a half-inactivation potential and a Boltzmann factor of -29.1 +/- 0.7 mV and 4.5 +/- 0.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of Ito recorded from native rat ventricular cells were exponentials with time constants of 213.2 +/- 4.1 msec and 23. +/- 1.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.54 +/- 0.13 mM and 0.14 +/- 0.02 mM on Kv4.3 and Ito, respectively. In both Kv4.3 and Ito, 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogeneous. CONCLUSION: The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac Ito current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogeneous, the blockade of this channel by specific drugs may not alter the normal heterogeneity of Ito current. (+info)
Properties and expression of Ca2+-activated K+ channels in H9c2 cells derived from rat ventricle.
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H9c2 is a clonal myogenic cell line derived from embryonic rat ventricle that can serve as a surrogate for cardiac or skeletal muscle in vitro. Using whole cell clamp with H9c2 myotubes, we observed that depolarizing pulses activated slow outward K+ currents and then slow tail currents. The K+ currents were abolished in a Ca2+-free external solution, indicating that they were Ca2+-activated K+ currents. They were blocked by apamin, a small-conductance Ca2+-activated K+ (SK) channel antagonist (IC50 = 6.2 nM), and by d-tubocurarine (IC50 = 49.4 microM). Activation of SK channels exhibited a bell-shaped voltage dependence that paralleled the current-voltage relation for L-type Ca2+ currents (ICa,L). ICa,L exhibited a slow time course similar to skeletal ICa, L, were unaffected by apamin, and were only slightly depressed by d-tubocurarine. RT-PCR analysis of the mRNAs revealed that rSK3, but not rSK1 or rSK2, was expressed in H9c2 myotubes but not in myoblasts. These results suggest that rSK3 channels are expressed in H9c2 myotubes and are primarily activated by ICa,L directly or indirectly via Ca2+-induced Ca2+ release from sarcoplasmic reticulum. (+info)
Vasorelaxing action of rutaecarpine: effects of rutaecarpine on calcium channel activities in vascular endothelial and smooth muscle cells.
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Rutaecarpine (Rut) has been shown to induce hypotension and vasorelaxation. In vitro studies indicated that the vasorelaxant effect of Rut was largely endothelium-dependent. We previously reported that Rut increased intracellular Ca2+ concentrations ([Ca2+]i) in cultured rat endothelial cells (ECs) and decreased [Ca2+]i in cultured rat vascular smooth muscle (VSMCs) cells. The present results showed that the hypotensive effect of Rut (10-100 microgram/kg i.v.) was significantly blocked by the nitric oxide synthase inhibitor Nomega-nitro-L-arginine. In aortic rings, Rut (0. 1-3.0 microM)-induced vasorelaxation was inhibited by Nomega-nitro-L-arginine and hydroquinone but not by antagonists of the various K+ channels, 4-aminopyridine, apamin, charybdotoxin, or glibenclamide. Rut (0.1 and 1.0 microM) inhibited the norepinephrine-induced contraction generated by Ca2+ influx and at 1.0 microM increased cyclic GMP (cGMP) production in endothelium-intact rings and to a lesser extent in endothelium-denuded rings. In whole-cell patch-clamp recording, nonvoltage-dependent Ca2+ channels were recorded in ECs and Rut (0.1, 1.0 microM) elicited an opening of such channels. However, in VSMCs, Rut (10.0 microM) inhibited significantly the L-type voltage-dependent Ca2+ channels. In ECs cells, Rut (1.0, 10.0 microM) increased nitric oxide release in a Ca2+-dependent manner. Taken together, the results suggested that Rut lowered blood pressure by mainly activating the endothelial Ca2+-nitric oxide-cGMP pathway to reduce smooth muscle tone. Although the contribution seemed to be minor in nature, inhibition of contractile response in VSMCs, as evidenced by inhibition of Ca2+ currents, was also involved. Potassium channels, on the other hand, had no apparent roles. (+info)