Biomedical subjects

D E Logothetis

Publications and source records attributed to D E Logothetis.

29 records · Page 2Linked to original sources

PubMed1995

Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms.

Within the mammalian hypothalamus, the suprachiasmatic nucleus (SCN) contains a circadian clock for timing of diverse neuronal, endocrine, and behavioral rhythms. By culturing cells from neonatal rat SCN on fixed microelectrode arrays, we have been able to record spontaneous action potentials from individual SCN neurons for days or weeks, revealing prominent circadian rhythms in firing rate. Despite abundant functional synapses, circadian rhythms expressed by neurons in the same culture are not synchronized. After reversible blockade of neuronal firing lasting 2.5 days, circadian firing rhythms re-emerge with unaltered phases. These data suggest that the SCN contains a large population of autonomous, single-cell circadian oscillators, and that synapses formed in vitro are neither necessary for operation of these oscillators nor sufficient for synchronizing them.

Action Potentials↗

PubMed1994

4-Aminopyridine binding and slow inactivation are mutually exclusive in rat Kv1.1 and Shaker potassium channels.

In the present study we have used two-electrode voltage-clamping of Xenopus oocytes expressing either Kv1.1 or Shaker B (ShB) delta 6-46 K+ channels to examine the effects of 4-aminopyridine (4-AP) on the process of slow inactivation. Neither of these channels exhibits fast inactivation. Channel activation was required for block by 4-AP in both channel types. In the absence of drug, inactivation of Kv1.1 and ShB delta 6-46 channels at 0 mV was biexponential [tau fast = 7.8 +/- 0.3 sec, tau slow = 33.9 +/- 0.9 sec, and Aslow/(Afast + Aslow) = 0.79 +/- 0.02 (n = 10) for Kv1.1 and tau fast = 3.5 +/- 0.4 sec, tau slow = 13.1 +/- 1.8 sec, and Aslow/(Afast + Aslow) = 0.35 +/- 0.06 (n = 3) for ShB delta 6-46]. In the presence of 4-AP, the rates of inactivation of Kv1.1 and ShB delta 6-46 were markedly slowed, resulting in a crossover phenomenon where, in the presence of drug, the outward current was smaller than control at the beginning of the depolarizing pulse but crossed over during the pulse to become larger than the control. The most obvious change induced by 0.2 mM 4-AP was a 2-fold slowing of the slow phase of inactivation [tau fast = 3.9 +/- 1.1 sec, tau slow = 67.1 +/- 3.6 sec, and Aslow/(Afast + Aslow) = 0.85 +/- 0.04 (n = 4) for Kv1.1 and tau fast = 3.5 +/- 0.4 sec, tau slow = 23.7 +/- 2.6 sec, and Aslow/(Afast + Aslow) = 0.75 +/- 0.02 (n = 3) for ShB delta 6-46, in the presence of 0.2 mM 4-AP]. In addition, there was a significant increase in the contribution of the slower phase of inactivation of ShB delta 6-46 channels in the presence of 4-AP. The slowed inactivation in the presence of 4-AP was accompanied by removal of 4-AP block. These results are consistent with the processes of 4-AP block and slow inactivation of Kv1.1 and ShB delta 6-46 channels being mutually exclusive.

4-Aminopyridine↗

PubMed1994

Aminopyridine block of Kv1.1 potassium channels expressed in mammalian cells and Xenopus oocytes.

The mechanism by which aminopyridines (APs) block cloned Kv1.1 K+ channels expressed either in the mammalian Sol-8 muscle cell line or in Xenopus oocytes was investigated using whole-cell patch-clamp and two-electrode voltage-clamp techniques. When Sol-8 cells were exposed to 4-AP (30 microM to 1 mM) or 3-AP (300 microM to 10 mM) for 1-2 min at a holding potential of -80 mV, delayed rectifier K+ currents activated by the first depolarization to +40 mV showed a small reduction in peak amplitude but exhibited a rapid decay phase that was absent in control records. However, currents elicited by subsequent pulses showed maximal block throughout the pulse. These results suggest that 4-AP requires the channel to be activated before block can occur readily. After a 10-min washout of 4-AP in the absence of channel activation, the current elicited at the beginning of the first depolarizing pulse was similar to that observed during maximal block. However, during the pulse the current increased in an exponential manner, to an amplitude similar to that seen in the absence of 4-AP. Subsequent pulses elicited currents with profiles similar to that observed in controls. These results suggest that channel activation is also required for unblock (i.e., 4-AP is trapped when the channel closes). Block of Kv1.1 channels by 4-AP was voltage dependent. The magnitude of block decreased progressively as the membrane potential was depolarized to voltages more positive than -20 mV. The concentration producing half-maximal inhibition (IC50) of Kv1.1 currents in Sol-8 cells at +40 mV at physiological pH (7.2) was 89 microM for 4-AP (pKa = 9.2) and 2.2 mM for 3-AP (pKa = 6.0). However, when the intracellular pH was lowered to 6.0 the IC50 for 3-AP decreased to 290 microM. These results are consistent with the charged form of the APs being the active species.

Aminopyridines↗

PubMed1993

Gating charge differences between two voltage-gated K+ channels are due to the specific charge content of their respective S4 regions.

Voltage-gated ion channels that differ in their primary amino acid sequence in the putative voltage sensor, the S4 region, show distinct voltage-sensing characteristics. In this study, we directly compared two voltage-gated K+ channels, the mammalian RCK1 with the Drosophila Shab11, and correlated the specific amino acid content of their respective S4 regions with the distinct voltage-sensing properties they exhibit. We find that specific differences in the charge content of the S4 region are sufficient to account for the distinct gating valence of each channel. However, differences in residues inside the S4 region are not sufficient to account for each channel's characteristic voltage range of activation.

Amino Acid Sequence↗

PubMed1992

Incremental reductions of positive charge within the S4 region of a voltage-gated K+ channel result in corresponding decreases in gating charge.

The S4 region of voltage-dependent ion channels is involved in the voltage-sensing mechanism of channel activation. Previous studies in fast inactivating channels have used non-steady-state measurements and thus have not allowed the quantitative assessment of activation parameters. Using site-directed mutagenesis and voltage-clamp recordings in a noninactivating channel (RCK1), we demonstrate that stepwise reductions of positive charge within the S4 region correlate with a progressive decrease in the channel's overall gating valence. In addition to testing for electrostatic behavior of individual charged residues, our study was designed to probe nonelectrostatic influences on charge movement. We provide evidence that individual charged residues behave differentially in response to the electric field, so that purely electrostatic influences cannot fully account for the gating movement of certain charges.

Amino Acid Sequence↗

PubMed1990

Rats anticipate and discriminate between two daily feeding times.

Intact rats and rats bearing lesions of the suprachiasmatic nuclei (SCNX rats) were trained to obtain food by pressing either of two levers located on opposite sides of a cylindrical cage. Intact rats were maintained in constant light (LL) and under daily light-dark (LD) cycles, SCNX rats in LL only. A restricted daily feeding schedule was next imposed, such that pressing one lever provided food for a limited duration (1 or 2 hr) at one time of day while pressing the second lever provided food for the same duration at another time of day. Most rats generally showed anticipatory lever-pressing preceding both daily feeding times, and several discriminated between the two, pressing the lever appropriate for each feeding time more than the inappropriate lever. Discrimination performance was better in intact rats in LD than in intact or SCNX rats in LL. These results indicate that rats can associate different food locations with different times of day, an ability previously known only in honeybees and birds.

Animals↗

PubMed1990

Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells.

We have cloned a cDNA coding for a delayed rectifier K+ channel from rat brain (RCK1) and rat muscle (RMK1) and expressed it in Xenopus oocytes and in a myoblast cell line (Sol-8). Stably transfected Sol-8 cells exhibited large outward K+ currents, which were indistinguishable from the K+ currents induced in Xenopus oocytes by injection of mRNA transcribed in vitro. RCK1 encodes a K+ channel with a unitary conductance of approximately 14 pS. The steep voltage dependence of channel opening resides in transitions between closed states, whereas the direct transitions into and out of the open state are very rapid and not markedly voltage-dependent. Channel inactivation is very slow, voltage-independent, and occurs from the open state only. We present a simple model that incorporates our findings and is consistent with the presumed structural symmetry of a functional K+ channel.

Animals↗

PubMed1989

Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons.

The major component of whole-cell Ca2+ current in differentiated, neuron-like rat pheochromocytoma (PC12) cells and sympathetic neurons is carried by dihydropyridine-insensitive, high-threshold-activated N-type Ca2+ channels. We show that these channels have unitary properties distinct from those of previously described Ca2+ channels and contribute both slowly inactivating and large sustained components of whole-cell current. The N-type Ca2+ currents are modulated by GTP binding proteins. The snail toxin omega-conotoxin reveals two pharmacological components of N-type currents, one blocked irreversibly and one inhibited reversibly. Contrary to previous reports, neuronal L-type channels are insensitive to omega-conotoxin. N-type Ca2+ channels appear to be specific for neuronal cells, since their functional expression is greatly enhanced by nerve growth factor.

Acetylcholine↗

PubMed1988

Specificity of action of guanine nucleotide-binding regulatory protein subunits on the cardiac muscarinic K+ channel.

The cardiac muscarinic receptor stimulates a potassium-selective ionic current (IK.ACh) through activation of a guanine nucleotide-binding regulatory protein. Purified alpha and beta gamma subunits of the guanine nucleotide-binding regulatory protein have each been reported to open the K+ channel. We have reported that nanomolar concentrations of purified brain beta gamma subunits activated IK.ACh in chicken embryonic atrial patches. In contrast, J. Codina, A. Yatani, D. Grenet, A.M. Brown, and L. Birnbaumer [(1987) Science 236, 442-445] subsequently reported that picomolar concentrations of activated erythrocyte alpha subunits (i.e., the 40-kDa alpha subunit that the authors call alpha K) opened K+ channels in guinea pig atrial patches. In this paper, we further explore the specificity of various beta gamma and alpha subunits in embryonic chicken and neonatal rat atrial patches. Beta gamma subunits from either human placenta (beta 35 gamma) or bovine brain (beta 35,36 gamma) activated IK.ACh whereas transducin beta gamma (beta 36 gamma) did not. The beta gamma activation was consistent in rat and chicken patches [118 of 123 patches (97%)]. Beta gamma subunits opened K+ channels at concentrations greater than or equal to 200 pM and maximally activated the channel at 10 nM. Beta gamma or guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) channel activation could be reversed by alpha 41-GDP. The purified brain beta gamma preparation was contaminated with less than 0.01% unactivated alpha. The detergent (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS), used to suspend the hydrophobic beta gamma, did not activate IK.ACh alone, with buffer, with heat-inactivated beta gamma, or with transducin beta gamma. Unactivated alpha subunits did not open K+ channels. Activated, alpha subunits purified from human erythrocytes (alpha 40-GTP[gamma-S]) or bovine brain (alpha 39-GTP[gamma-S]) at concentrations of 10 pM or higher (up to 1 nM) opened K+ channels less frequently in chicken atrial patches [5 of 27 patches (19%) and 9 of 35 patches (26%), respectively] than in rat atrial patches [5 of 11 patches (45%) and 11 of 19 patches (58%), respectively]. Negative results were not due to patch vesicle formation. Other experiments indicated that alpha and beta gamma activated the same population of channels. Activation of the channel by both beta gamma and alpha subunits implies a more complicated scheme for guanine nucleotide-binding regulatory protein action than previously proposed.

Animals↗

PubMed1988

Delayed rectifier K+ current in embryonic chick heart ventricle.

Single ventricular cells from 7- to 10-day-old embryonic chicks were studied with the use of whole cell, outside-out, and cell-attached patch-clamp techniques. The macroscopic delayed rectifier current, IK, activated at membrane potentials above -25 mV. Peak IK at +40 mV was 103 +/- 20 pA (5.7 +/- 1 microA/cm2). IK was selective for K ions with reversal potentials close to the Nernst equilibrium potentials. The onset of current during a voltage step was sigmoidal and was fit by the function IK = IKoo (1-e-t/tau)2. The peak time constant of activation was greater than 6 s at -25 mV (22 degrees C). Significant inactivation was not observed. IK was blocked by intracellular cesium, extracellular 1 mM 4-aminopyridine, 20 mM tetraethylammonium chloride, and 1 mM barium chloride. Single-channel recordings revealed a K+-selective channel with a slope conductance of 15 pS (extracellular [K+] = 4 mM, intracellular [K+] = 145 mM). An ensemble average of consecutive single-channel traces reproduced the whole cell current. The single-channel density was approximately 0.04/micron 2 based on frequency of patches containing the 15-pS single conductance. Approximately 100 channels/cell would sum to account for the net IK. We have described for the first time a channel underlying the main delayed rectifier current and, as such, a main repolarization current in chick ventricle.

4-Aminopyridine↗

PubMedDate not supplied

The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart.

Subunits of guanine nucleotide regulatory proteins purified from bovine cerebral cortex were used to perfuse the intracellular surface of excised patches of chick embryonic atrial cells. Single-channel current measurements unexpectedly indicate that the beta gamma, and not the alpha subunits, are responsible for activating the muscarinic-gated potassium channel.

Animals↗