| Intracellular Calcium Directly Inhibits Potassium M Channels in Excised Membrane Patches from Rat Sympathetic Neurons Neuron, Volume 16, Issue 1, 1 January 1996, Pages 151-162 A.A Selyanko and D.A Brown Summary Complex effects of altering intracellular [Ca] on M-type K currents have previously been reported using whole-cell current recording. To study the direct effect of Ca on M-channel activity, we have applied Ca to the inside face of membrane patches excised from rat superior cervical sympathetic ganglion cells. Ca rapidly and reversibly inhibited M-channel activity in 28/44 patches by up to 87%, with a mean IC of 100 nM. This effect persisted in the absence of ATP, implying that it was not due to phosphorylation/dephosphorylation. A similar effect was observed in 13/13 cell-attached patches when cells were transiently “Ca-loaded” by adding 2 mM Ca to a 25 mM K solution bathing the extrapatch cell membrane. These observations provide new evidence that Ca can directly inhibit M channels, so supporting the view that Ca might mediate M current inhibition following muscarinic receptor activation. Summary | Full Text | PDF (301 kb) |
| Ca- and Voltage-Dependent Gating of Ca- and ATP-Sensitive Cationic Channels in Brain Capillary Endothelium Biophysical Journal, Volume 85, Issue 1, 1 July 2003, Pages 313-327 László Csanády and Vera Adam-Vizi Abstract Biophysical properties of the Ca-activated nonselective cation channel expressed in brain capillaries were studied in inside-out patches from primary cultures of rat brain microvascular endothelial cells. At −40mV membrane potential, open probability () was activated by cytosolic [Ca]>1M and was half-maximal at ∼20M. Increasing [Ca] stimulated opening rate with little effect on closing rate. At constant [Ca], was voltage-dependent, and effective gating charge corresponded to 0.6±0.1 unitary charges. Depolarization accelerated opening and slowed closing, thereby increasing apparent affinity for Ca. Within ∼1min of excision, declined to a lower steady state with decreased sensitivity toward activating Ca when studied at a fixed voltage, and toward activating voltage when studied at a fixed [Ca]. Deactivated channels opened ∼5-fold slower and closed ∼10-fold faster. The sulfhydryl-reducing agent dithiotreitol (1mM) completely reversed acceleration of closing rate but failed to recover opening rate. Single-channel gating was complex; distributions of open and closed dwell times contained at least four and five exponential components, respectively. The longest component of the closed-time distribution was markedly sensitive to both [Ca] and voltage. We conclude that the biophysical properties of gating of this channel are remarkably similar to those of large-conductance Ca-activated K channels. Abstract | Full Text | PDF (427 kb) |
| Calcineurin Regulates M Channel Modal Gating in Sympathetic Neurons Neuron, Volume 16, Issue 1, 1 January 1996, Pages 163-173 Neil V Marrion Summary The M current regulates neuronal excitability, with its amplitude resulting from high open probability modal M channel behavior. The M current is affected by changing intracellular calcium levels. It is proposed that internal calcium acts by regulating M channel modal gating. Intracellular application of a preactivated form of the calcium-dependent phosphatase calcineurin (CaN) inhibited the macroscopic M current, while its application to excised inside-out patches reduced high open probability M channel activity. Addition of ATP reversed the action of CaN on excised patches. The change in M channel gating induced by CaN was different from the effect of muscarine. A kinetic model supports the proposition that shifts in channel gating induced by calcium-dependent phosphorylation and dephosphorylation control M current amplitude. Summary | Full Text | PDF (247 kb) |
Copyright © 1995 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 69, Issue 6, 2364-2368, 1 December 1995
doi:10.1016/S0006-3495(95)80105-0
Research Article
L. Goldman
Department of Physiology, School of Medicine, University of Maryland, Baltimore 21201, USA.
Na channel gating parameters in a number of preparations are translated along the voltage axis in excised patches compared to cell attached or whole cell recording. The aim of this study is to determine whether these changes in gating behavior continue over an extended period or, rather, develop rapidly on excision with stationary kinetics thereafter. Average currents were constructed from single-channel records from neuroblastoma N1E 115 at various times after excision, excluding the first 5min, in eight inside-out excised patches. Single exponentials were fitted to the current decay of the average records, and the mean time constant for each patch was determined. Values were plotted as the percentage difference from these means for each patch against time from excision. Collected results show no obvious trend in values from 5min to 2 h. Kinetics are stationary, and shifts in Na channel gating parameters along the voltage axis seen in excised as compared to whole cell configuration in neuroblastoma must be complete by the first few minutes after excision. Raising the internal Na concentration reduced the single channel current amplitude, confirming that these are Na channels.