Number Of Channel Openings Research Articles

Direct Effects of Calmodulin on NMDA Receptor Single-Channel Gating in Rat Hippocampal Granule Cells

NMDA receptors are glutamate-sensitive ion channel receptors that mediate excitatory synaptic transmission and are widely implicated in synaptic plasticity and integration of synaptic activity in the CNS. This is in part attributable to the high calcium permeability of the ion channel, which allows receptor activation to influence the intracellular calcium concentration and also the slow time course of NMDA receptor-mediated synaptic currents. NMDA receptor activity is also regulated by the intracellular calcium concentration through activation of various calcium-dependent proteins, including calmodulin, calcineurin, protein kinase C, and alpha-actinin-2. Here, we have shown that calmodulin reduces the duration of native NMDA receptor single-channel openings from 3.5 +/- 0.6 msec to 1.71 +/- 0.2 msec in agreement with previous studies on recombinant NMDA receptors (Ehlers et al., 1996). NMDA receptor single-channel amplitudes and shut times were not affected. However, calmodulin reduced the duration of groups of channel openings called superclusters, which determine the slow time course of synaptic currents, from 121 +/- 25.4 msec to 60.4 +/- 11.6 msec. In addition, total open time, number of channel openings, and charge transfer per supercluster were all reduced by calmodulin. A 68% decrease in charge transfer per supercluster suggests that calmodulin activation will significantly reduce calcium influx during synaptic transmission. These results suggest that calmodulin-dependent inhibition of NMDA receptors will reduce the amplitude and time course of excitatory synaptic currents and thus affect synaptic plasticity and integration of synaptic activity in the CNS.

Modulation of L-type Ca 2+ channels in UMR 106 cells by parathyroid hormone-related protein

The effects of rat parathyroid hormone-related protein (rPTHrP) and bovine and rat parathyroid hormone (bPTH and rPTH) on L-type Ca 2+ channels in UMR 106 cells were investigated using the patch clamp technique. rPTHrP increased the whole cell L-type Ca 2+ channel currents and the increase was concentration dependent. rPTHrP, at a concentration of 62.5 nM, increased the L-type Ca 2+ channel current by 122±25%. bPTH was less potent. A concentration of 7.5 μM bPTH increased the current by 99±24%. Results obtained with rPTH were similar to those obtained using bPTH. Single channel measurements, using the cell-attached version of the patch clamp technique, showed an increase in both the number of channel openings and the mean open time when the cells were exposed to rPTHrP. This suggested that rPTHrP affected the gating of L-type Ca 2+ channels in UMR 106 cells. This study demonstrates that the actions of bPTH and rPTHrP in UMR cells are mediated in part by extracellular Ca 2+ entry. PTHrP, a paracrine agent important in development, is more potent in regulating Ca 2+ entry than PTH.

Patch clamp studies of the Thr1313met mutant sodium channel causing paramyotonia congenita

Paramyotonia congenita (PC) is an autosomal-dominant disorder due to a point mutation in the adult skeletal muscle Na channel gene. Muscle fibers from PC patients have normal membrane properties at 32°C. At 27°C, they are inexcitable, have increased Na conductance, and have a reduced resting membrane potential of −40 mV. To define the biophysical basis for the muscle membrane abnormalities, we performed patch clamp whole-cell and outside-out single Na channel studies at 22°C on cultured human muscle cells from 4 control patients and 2 sisters with PC and the thr1313met mutant Na channel. The whole-cell studies showed no difference in window currents. Unlike cells transfected with the thr1313met mutant Na channel, the inactivation time constant, τh, for PC cells was similar to control cells. For PC recordings containing long-duration single Na channel openings, mean open time was prolonged at −60, −40, and −20 mV. The long-duration Na channel openings occurred randomly with no evidence of modal gating. The number of channel openings, occurrence of late openings, and the prolonged mean open time resulted in a sustained inward Na current at −40 mV. We suggest that the biophysical marker of the thr1313met mutant Na channel is a voltage- and temperature-dependent abnormality in mutant single Na channel behavior. © 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 1736–1747, 2000.

Patch clamp studies of the thr1313met mutant sodium channel causing paramyotonia congenita.

Paramyotonia congenita (PC) is an autosomal-dominant disorder due to a point mutation in the adult skeletal muscle Na channel gene. Muscle fibers from PC patients have normal membrane properties at 32 degrees C. At 27 degrees C, they are inexcitable, have increased Na conductance, and have a reduced resting membrane potential of -40 mV. To define the biophysical basis for the muscle membrane abnormalities, we performed patch clamp whole-cell and outside-out single Na channel studies at 22 degrees C on cultured human muscle cells from 4 control patients and 2 sisters with PC and the thr1313met mutant Na channel. The whole-cell studies showed no difference in window currents. Unlike cells transfected with the thr1313met mutant Na channel, the inactivation time constant, tau(h), for PC cells was similar to control cells. For PC recordings containing long-duration single Na channel openings, mean open time was prolonged at -60, -40, and -20 mV. The long-duration Na channel openings occurred randomly with no evidence of modal gating. The number of channel openings, occurrence of late openings, and the prolonged mean open time resulted in a sustained inward Na current at -40 mV. We suggest that the biophysical marker of the thr1313met mutant Na channel is a voltage- and temperature-dependent abnormality in mutant single Na channel behavior.

Arginine Vasopressin–Induced Potentiation of Unitary L-Type Ca 2+ Channel Current in Guinea Pig Ventricular Myocytes

The effects of arginine vasopressin (AVP) on L-type Ca2+ channels were studied by recording single-channel activity from cell-attached patches on isolated guinea pig ventricular myocytes, with 100 mmol/L Ba2+ used as the charge carrier. Bath application of AVP (100 nmol/L) reversibly increased channel open probability by a factor of 2.92 +/- 1.43 (n = 15) because of the increased number of channel openings and increased open times. AVP did not change the amplitudes of single-channel currents (1.17 +/- 0.10 pA in the control condition and 1.12 +/- 0.11 pA after AVP, at +20 mV; n = 6). In our experimental conditions, in which myocytes were bathed in Ca(2+)-free high-potassium solutions, AVP-induced potentiation was observed without changes in [Ca2+]i measured by fura 2 fluorescence signals (estimated [Ca2+]i, approximately 80 nmol/L). The AVP-induced increase in channel open probability was abolished by OPC-21268 (8 mumol/L), a specific blocker of V1 receptor, but not by a V2 blocker, OPC-31260 (5 mumol/L). AVP-induced potentiation was also suppressed by a broad-spectrum protein kinase inhibitor, H7 (100 mumol/L, bath application), but not by H89 (1 mumol/L), a blocker with high specificity to protein kinase A. AVP application after the treatment by phorbol ester (phorbol 12-myristate 13-acetate, 100 nmol/L for 1 hour) failed to potentiate the channel activity. These results raised the possibility that protein kinase C might be involved during signal transduction. Our results provide direct evidence that AVP potentiates cardiac L-type Ca2+ currents via V1 receptor stimulation.

Activation and inactivation of the bursting potassium channel from fused Torpedo synaptosomes.

1. The voltage dependence of the bursting potassium channel in fused synaptosomes from Torpedo electric organ was studied in vitro, using the inside-out and the cell-attached configurations of the patch clamp technique. 2. The patch of membrane was held at various holding potentials (-140 to -50 mV) and then stepped to test potentials (-50 to +40 mV) for periods ranging from 5 to 300 ms. Each potential step was repeated 200-600 times. After subtraction of the capacitative transients and the leakage currents, an ensemble-averaged current was obtained. This ensemble current showed a marked activation upon depolarization, followed by an inactivation. 3. The activation of the bursting potassium channel is markedly dependent on the voltage step. Activation was detected at voltages positive to -50 mV. The peak of the ensemble current increases with the degree of depolarization, while the time to the peak decreases. With progressively larger depolarization, there is a shortening in the delay between the onset of the voltage step and the opening of the bursting potassium channels. 4. The inactivation phase of the ensemble current could be described adequately in most of the experiments, as a single exponential decay to a steady-state inactivation level. The time constant of inactivation was not markedly voltage dependent. 5. Single channel analysis of the inactivation reveals that it is due to a reduction in the number of channel openings and not due to changes in single channel current amplitude or channel mean open time along the pulse. 6. The holding potential has a marked effect on the peak amplitude of the ensemble current, indicating that hyperpolarization removes inactivation and depolarization induces it. The peak amplitude vs. voltage relation was fitted by the Boltzmann equation. The half-maximal inactivation was -105.2 +/- 5.8 mV (mean +/- S.E.M.), suggesting that at the resting potential a substantial fraction of the bursting potassium channels is in an inactivated state. 7. Two-pulse experiments show that the recovery from inactivation is a slow process which lasts well over 1 s. 8. High-frequency stimulation (20-66.7 Hz) by 5 ms pulses produces a progressive decline in the peak ensemble current amplitude. The decline is larger at higher stimulation frequencies.(ABSTRACT TRUNCATED AT 400 WORDS)

Sodium channel inactivation from resting states in guinea-pig ventricular myocytes.

1. Unitary Na+ channel currents were recorded from isolated guinea-pig ventricular myocytes using the cell-attached patch-clamp technique with high [Na+] in the pipette to enhance the signal-to-noise ratio. 2. The probability that the channel enters the inactivated state (I) directly from resting states (C) was investigated over a wide range of membrane potentials. 3. At membrane potentials of -60 mV or more positive, Markov chain theory was used to estimate the probability of C----I from histograms of the number of channel openings per depolarizing period. Holding potentials at least as negative as -136 were required to ensure that all channels resided in C prior to depolarization. 4. At membrane potentials negative to -60 mV, a two-pulse protocol was employed to determine the probability of C----I from the fraction of blank sweeps during the pre-pulse with correction for missed events. 5. The probability of C----I was found to be steeply voltage dependent at negative potentials, falling from 0.87 +/- 0.03 (mean +/- S.D.) at -91 mV to 0.42 +/- 0.01 at -76 mV. At potentials positive to -60 mV, this probability was less steeply voltage dependent and decayed to near zero at 0 mV. 6. Under physiological conditions, C----I transitions may produce appreciable Na+ channel inactivation at diastolic potentials. At potentials above the action potential threshold, inactivation is much more likely to occur from the open state.

Modulation of the N-methyl-D-aspartate channel by extracellular H+.

The influence of external [H+] on whole-cell and single-channel currents activated by glutamate agonists was studied in rat hippocampal neurons. In the pH range between 6.6 and 8.0, changes in external [H+] had negligible influence on the amplitude and kinetics of the monovalent ion-carrying currents activated by the agonists quisqualate and kainate. The divalent ion-carrying N-methyl-D-aspartate (NMDA)-activated current, on the other hand, was strongly modulated by extracellular [H+]. Increased external [H+] suppressed, whereas decreased external [H+] enhanced, the NMDA-activated current. Changes in internal [H+] had little or no effect on the NMDA-activated current. Modulation of the NMDA-activated current resulted primarily from changes in the number of channel openings. Neither the unitary conductance nor the individual open dwell-times were significantly affected. These results suggest that the protonation site is on the external aspect of the channel and is far removed from the channel permeation pathway. Because interactions between H+, NMDA, and glycine in activating the current were predominantly noncompetitive, our results suggest that the modulatory effect of H+ was not associated with changes in receptor-agonist affinities. These results suggest that modulation of the NMDA-receptor channel by [H+] may be an intrinsic protective mechanism by which calcium influx into neurons is regulated, particularly in hypoxic/ischemic conditions.

Single-channel analysis of the potassium permeability in HeLa cancer cells: Evidence for a calcium-activated potassium channel of small unitary conductance

Cell-attached and inside-out patch-clamp experiments (O.P. Hamill et al., Pfluegers Arch. 391:85-100, 1981) were undertaken in order to characterize the molecular mechanisms responsible for the calcium-dependent potassium permeability observed in HeLa cancer cells. Our result essentially indicate that the HeLa cell external membrane contains potassium channels whose activity can be triggered within an internal calcium concentration range of 0.1 to 1 microM. This particular channel was found to behave as an inward rectifier in symmetrical 200 mM KCl with a conductance of 50 and 10 pS at large negative and large positive membrane potentials, respectively. I/V curves were also measured in 10, 20, 75, 200 and 300 mM KCl and the data interpreted in terms of a one-site-two-barrier model. The channel activity appeared to be nearly voltage independent within the voltage range -100 to +100 mV, an increase of Po, the open channel probability, being observed at large negative potentials only. In addition, the results obtained from inside-out experiments on the relationship between Po and the cytoplasmic free-calcium concentration have led to conclude that four calcium ions are probably required in order to open the channel. In this regard it was found that an increase of the internal free-calcium level affects more the number of channel openings per second than the actual channel mean lifetime. Finally, it is concluded following a time interval distribution analysis, that this particular channel has at least three closed states and two open states.

Interaction between sodium channels in mouse neuroblastoma cells

Single sodium channels in mouse neuroblastoma cells (N1E 115) were studied in cell-attached patches. During a series of consecutive responses to depolarizing pulses, records with and without channel opening were seen to form clusters rather than appearing randomly. The probability of finding open channels on a record seemed to increase with increasing number of channel openings. The open times of channels became shorter with increasing closed time interval measured between consecutive channel openings. Overlapping openings showed a voltage-dependent open time, in contrast to single openings which had voltage-independent open time. On the basis of these observations interaction between neighbouring sodium channels is suggested.