Perforated Whole-cell Configuration Research Articles

Perforated Whole-Cell Recordings in Automated Patch Clamp Electrophysiology.

The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical access to the cells are obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca2+), prevents channel current "run down," and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.

Cell coupling in mouse pancreatic β-cells measured in intact islets of Langerhans

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell (due to the firing of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell KATP channel conductance (GK,ATP) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC50 of approximately 4mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca2+]i waves to spread with a speed of approximately 80 microms-1, similar to that observed experimentally in confocal [Ca2+]i imaging.

The inhibitors of protein acylation, cerulenin and tunicamycin, increase voltage-dependent Ca 2+ currents in the insulin-secreting INS 832/13 cell

As it has been suggested that protein acylation plays a role in nutrient stimulus–secretion coupling in the pancreatic β-cell, we examined the insulin-secreting INS 832/13 β-cell line for evidence that protein acylation was involved. The perforated whole-cell configuration was employed to voltage-clamp INS 832/13 cells. Voltage pulses were applied and Ca 2+ currents measured in the presence and absence of the protein acylation inhibitors cerulenin and tunicamycin. Both inhibitors enhanced the peak amplitude of I Ca,L. Both increased the peak inward current in the range between −40 and +30 mV and shifted the apparent maximum current by 10 mV in the hyperpolarizing direction without affecting the activation threshold of −40 mV. The two drugs had qualitatively and quantitatively similar effects. Steady-state activation curves revealed that cerulenin and tunicamycin shifted the activation curves in the hyperpolarization direction. Activation time constants were significantly reduced in the presence of both drugs. The Ca 2+ charge influx was increased by the drugs at all potentials tested. In contrast to these effects on the L-type Ca 2+ channel, the two inhibitors of protein acylation had no effect on the ATP-sensitive K + channel. The results suggest that protein acylation exerts a tonic inhibitory effect on L-type Ca 2+ channel function in the insulin-secreting β-cell.

Pattern of Ca2+ increase determines the type of secretory mechanism activated in dog pancreatic duct epithelial cells

Intracellular calcium concentration ([Ca(2+)](i)) is a key factor controlling secretion from various cell types. We investigated how different patterns of [Ca(2+)](i) signals evoke salt secretion via ion transport mechanisms and mucin secretion via exocytosis in dog pancreatic duct epithelial cells (PDEC). Activation of epithelial P2Y(2) receptors by UTP generated two patterns of [Ca(2+)](i) change: 2-10 microm UTP induced [Ca(2+)](i) oscillations, whereas 100 microm UTP induced a sustained [Ca(2+)](i) increase, both in the micromolar range. As monitored by carbon-fibre amperometry, the sustained [Ca(2+)](i) increase stimulated a larger increase in exocytosis than [Ca(2+)](i) oscillations, despite their similar amplitude. In contrast, patch-clamp recordings revealed that [Ca(2+)](i) oscillations synchronously activated a K(+) current as efficiently as the sustained [Ca(2+)](i) increase. This K(+) current was mediated by intermediate-conductance Ca(2+)-activated K(+) channels (32 pS at -100 mV) which were sensitive to charybdotoxin and resistant to TEA. Activation of these Ca(2+)-dependent K(+) channels hyperpolarized the plasma membrane from a resting potential of -40 mV to -90 mV, as monitored in perforated whole-cell configuration, in turn enhancing Na(+)-independent, Cl(-)-dependent and DIDS-sensitive HCO(3)(-) secretion, as monitored through changes in intracellular pH. PDEC therefore encode concentrations of purinergic agonists as different patterns of [Ca(2+)](i) changes, which differentially stimulate K(+) channels, the Cl(-)-HCO(3)(-) exchanger, and exocytosis. Thus, in addition to amplitude, the temporal pattern of [Ca(2+)](i) increases is an important mechanism for transducing extracellular stimuli into different physiological effects.

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca(2+)-activated K(+) channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I(VNSCC)) and a relatively high-threshold voltage-activated (L-type) Ca(2+) current (I(L)). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I(VNSCC) or I(L) in dialyzed cells. ATP (1 mM) increased I(VNSCC) and decreased I(L) in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I(VNSCC). ADP decreased I(L) but had no effect on I(VNSCC). The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I(VNSCC). ATP stimulation of I(VNSCC) was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y(4) is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I(VNSCC) and depresses I(L) via binding of P2Y(4) receptors and stimulation of the phospholipase C/PKC pathway.

Intracellular Ca(2+) modulation of ATP-sensitive K(+) channel activity in acetylcholine-induced activation of rat pancreatic beta-cells.

We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic beta-cells. In an extracellular solution with 5.5 mM glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca(2+) channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic beta-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from -90 to -50 mV induced membrane currents mostly through ATP-sensitive K(+) channels (K(ATP)). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca(2+) chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through K(ATP) with 0.5 mM EGTA, but not with 10 mM EGTA, in the pipette solution. Intracellular application of GDPbetaS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca(2+) concentration inside the membrane from 10 nM-10 microM decreased K(ATP) activity only in the presence of ATP. The affinity of ATP to K(ATP) became 4.5 times higher with the higher concentration of Ca(2+). These results suggest that Ca(2+) from ACh receptor signaling modulates the sensitivity of K(ATP) to ATP. A positive-feedback mechanism of intracellular Ca(2+)-dependent Ca(2+) influx was also demonstrated.

Intracellular Ca2+ Modulation of ATP-Sensitive K+ Channel Activity in Acetylcholine-Induced Activation of Rat Pancreatic -Cells

We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic β-cells. In an extracellular solution with 5.5 mm glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca2+ channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic β-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from −90 to −50 mV induced membrane currents mostly through ATP-sensitive K+ channels (KATP). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca2+ chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through KATP with 0.5 mm EGTA, but not with 10 mm EGTA, in the pipette solution. Intracellular application of GDPβS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca2+ concentration inside the membrane from 10 nm–10 μm decreased KATP activity only in the presence of ATP. The affinity of ATP to KATP became 4.5 times higher with the higher concentration of Ca2+. These results suggest that Ca2+ from ACh receptor signaling modulates the sensitivity of KATP to ATP. A positive-feedback mechanism of intracellular Ca2+-dependent Ca2+ influx was also demonstrated.

Whole-cell patch-clamp: true perforated or spontaneous conventional recordings?

Perforated whole-cell patch-clamp recordings obtained with nystatin are frequently used to preserve intracellular integrity. However, the perforated-patch configuration may sometimes undergo a spontaneous change into the conventional whole-cell configuration, especially when lymphocytes are investigated. The electrophysiological criteria-- previously described--for establishing the existence of the perforated whole-cell configuration have been shown to be insufficient. Thus, the dye eosin, applied to the pipette solution, was tested as a tool for discriminating between the perforated and the conventional whole-cell configurations on rat T-lymphocytes. The dye never entered the cell from the pipette during the entire measurement in the perforated whole-cell configuration. In contrast, all cells in the conventional whole-cell configuration became red immediately after membrane rupture. Eosin barely changed the currents studied. The results suggest that eosin is a dye of choice for verifying a true perforated-patch configuration.

CAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic beta-cells.

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.

Evidence for an electrogenic Na+-HCO3- symport in rat cardiac myocytes.

1. The perforated whole-cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+-HCO3- cotransport in isolated rat ventricular myocytes. 2. Switching from Hepes buffer to HCO3- buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 +/- 0.4 mV (n = 9, P < 0.05). In the presence of HCO3-, the anion blocker SITS depolarized RMP by 2.6 +/- 0.5 mV (n = 5, P < 0.05). No HCO3--induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 +/- 6.1 % shorter in the presence of HCO3- than in its absence (n = 6, P < 0.05). 3. Quasi-steady-state currents were evoked by voltage-clamped ramps ranging from -130 to +30 mV, during 8 s. The development of a novel component of Na+-dependent and Cl--independent steady-state outward current was observed in the presence of HCO3-. The reversal potential (Erev) of the Na+-HCO3- cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3-:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole-cell experiments. Under these conditions, INa,Bic reversed at -96.4 +/- 1.9 mV (n = 5), being consistent with the influx of 2 HCO3- ions per Na+ ion through the Na+-HCO3- cotransporter. 4. In the presence of external HCO3-, after 10 min of depolarizing the membrane potential (Em) with 45 mM extracellular K+, a significant intracellular alkalinization was detected (0.09 +/- 0. 03 pH units; n = 5, P < 0.05). No changes in pHi were observed when the myocytes were pre-treated with the anion blocker DIDS (0.001 +/- 0.024 pH units; n = 5, n.s.), or when exposed to Na+-free solutions (0.003 +/- 0.037 pH units; n = 6, n.s.). 5. The above results allow us to conclude that the cardiac Na+-HCO3- cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.