Patch-clamp Single-channel Current Recording Research Articles

Biophysical and Pharmacological Properties of Large Conductance Ca2+-Activated K+ Channels in N1E-115 Cells

The large conductance Ca2+-activated K+ channels in differentiated mouse neuroblastoma N1E-115 cells have been studied using patch-clamp single-channel current recording in excised membrane patches. These channels displayed a unitary conductance of 200 pS under symmetrical K+ concentrations. Effects of blockade by TEA+, Cs+ and Ba2+ were different and argued for distinct action mechanisms. The open probability of these channels increased with increasing internal calcium and membrane potential. Maximum sensitivity of these channels ranged over physiological variations of internal calcium at membrane potentials close to zero, suggesting a physiological role for these channels in regulating the membrane potential and Ca2+ influx through voltage-dependent Ca2+ channels.

Effects of ADP upon the ATP-sensitive K+ channel in rat ventricular myocytes

The effects of ADP upon the gating of ATP-sensitive K+ channels from rat ventricular myocytes have been investigated by patch-clamp single-channel current recording experiments. ADP was applied to the internal surface of excised inside-out membrane patches and depending upon the experimental protocol and the concentration it was found that ADP could either inhibit or stimulate openings of ATP-sensitive K+ channels. In the absence of inactivation, ATP-sensitive K+ channels were inhibited by ADP in a dose-dependent manner. Partially inactivated channels, on the other hand, were stimulated by low (10 to 250 microM) and inhibited by high (greater than 250 microM) concentrations of ADP. ATP-sensitive K+ channels which were being inhibited by ATP (less than 1 mM) could be opened by the simultaneous application of ADP (50 microM to 1 mM). ADP had no effect upon channels inhibited by mM concentrations of ATP. The situation was further complicated when it was found that inhibition evoked by ADP was strongly attenuated by the presence of Mg2+ ions whilst channel stimulation, whether of partially inactivated channels or channels inhibited by ATP, required the presence of Mg2+ ions. The analog of ADP, ADP beta S, always evoked inhibition of ATP-sensitive K+ channels which was not affected by the presence or absence of Mg2+ ions.

Voltage-activation of high-conductance K + channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca 2+ concentration

Patch-clamp single-channel current recording experiments have been carried out on intact insulin-secreting RINm5F cells. Voltage-activation of high-conductance K + channels were studied by selectively depolarizing the electrically isolated patch membrane under conditions with normal Ca 2+ concentration in the bath solution but with or without Ca 2+ in the patch pipette solution. When Ca 2+ was present in the pipette, 40 mV to 120 mV depolarizing pulses (100 ms) from the normal resting potential (−70 mV) regularly evoked tetraethylammonium-sensitive large outward single-channel currents and the average open state probability during the pulses varied from about 0.015 (40 mV pulses) to 0.1 (120 mV pulses). In the absence of Ca 2+ in the pipette solution the same protocol resulted in fewer and shorter K + channel openings and the open-state probability varied from about 0.0015 (40 mV pulses) to about 0.03 (120 mV pulses). It is concluded that Ca 2+ entering voltage-gated channels raises [Ca 2+] i locally and thereby markedly enhances the open-state probability of tetraethylammonium-sensitive voltage-gated high-conductance K + channels.

ATP-sensitive K+ channels in an insulin-secreting cell line are inhibited byd-glyceraldehyde and activated by membrane permeabilization

The control of K+ channels in the insulin-secreting cell line RINm5F has been investigated by patch-clamp single-channel current recording experiments. The unitary current events recorded from cell-attached patches are due to large and small inwardly rectifying ATP-sensitive K+ channels with conductance properties similar to the two channels previously identified in primary cultured rat islet cells (Findlay, I., Dunne, M.J., & Petersen, O.H. J. Membrane Biol. 88:165-172, 1985). Cell permeabilization through brief exposure to 10 microM digitonin or 0.05% saponin (outside the isolated membrane patch area) results in a dramatic increase in current through the cell-attached patch due to opening of many large and small K+-selective channels. These channels are inhibited in a dose-dependent manner by ATP applied to the bath (near-complete inhibition by 5 mM ATP). During prolonged ATP exposure (1-5 min) the initial inhibition is followed by partial recovery of channel activity, although further activation does occur when ATP is subsequently removed. From the maximal number of coincident channel openings in the permeabilized cells (in the absence of ATP), it is estimated that there are on average 12 large ATP-sensitive K+ channels per membrane patch, but in the intact cells less than 5% of the membrane patches exhibited three or more coincident K+ channel openings, indicating the degree to which the channels are inhibited in the resting condition by endogenous ATP. Stimulation of RINm5F cells to secrete insulin was carried out by challenging intact cells with 10 mM D-glyceraldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+-and voltage activated K+ channel in apical cell membrane of gallbladder epithelium fromTriturus

The presence of Ca2+- and voltage-activated K+ channels was directly demonstrated in the apical cell membrane of gallbladder epithelium by patch-clamp single-channel current recording. In K+-depolarized epithelial cells, negative pipette potentials induced outward current steps when the patch-pipette was filled with Na+-rich solution and these current steps were not affected by the presence or absence of Cl-. When K+-rich solution was in the pipette and K+-depolarized cells were examined, the current-voltage relations were linear with a single-channel conductance of 140 pS and polarity was reversed at 0 mV. In excised inside-out membrane patches, raising the free Ca2+ concentration of the medium facing the inner side of the membrane from 10(-7) to 10(-6) M evoked a marked increase in open state probability of the channels without affecting the elementary current steps. This suggests that intracellular Ca2+ as a second messenger plays a crucial role in the regulatory mechanism of the membrane potential by modulating the high-conductance apical K+ channels.

Two types of cation channels in the basolateral cell membrane of human salivary gland acinar cells.

Giga-seal patch-clamp single-channel current recording was applied to the basolateral cell membrane of human salivary gland acinar cells. The results indicate that the presence of Ca2+- and voltage-activated K+-channel having a unit conductance of 160 pS and Ca2+-activated Na+ permeable channel, having a unit conductance of 15 pS.

Calcium-activated cation channel in rat thyroid follicular cells

Using the patch-clamp single-channel current recording technique, a cation channel in the contraluminal membrane of rat thyroid follicular cells has been characterized. The channel has a unit conductance of about 35 pS and is equally permeable to sodium and potassium. The pattern of channel opening and closing is independent of the membrane potential. The channel is only operational when the ionized calcium concentration in the fluid which is in contact with the inside of the membrane is at least 1 μM. This conductance pathway can be classified as a calcium dependent non-selective cation channel and could explain stimulant-evoked depolarizations in the thyroid follicular cells.

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium.

The Ca2+-activated K+ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na+ outside, K+ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than -40 mV. In symmetrical K+-rich solutions the single-channel I/V relationship was linear, the null potential was 0 mV and the single-channel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca2+-free solution containing 2mM EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca2+ concentration in contact with the membrane inside ( [Ca2+]i) to 1.5 X 10(-7) M had little effect on the relationship between membrane potential and P. When [Ca2+]i was increased to 3 X 10(-7) M and 6 X 10(-7) M smaller potential changes were required to open the channels. Increasing [Ca2+]i further to 8 X 10(-7) M again activated the channels, but the relationship between membrane potential and P was complex. Changing the membrane potential from -50 mV to +20 mV increased P from near 0 to 0.6 but further polarization to +50 mV decreased P to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca2+]i = 1 and 2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)