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

2008 ◽  
Vol 366 (1880) ◽  
pp. 3503-3523 ◽  
Author(s):  
Quan Zhang ◽  
Juris Galvanovskis ◽  
Fernando Abdulkader ◽  
Christopher J Partridge ◽  
Sven O Göpel ◽  
...  
Keyword(s):  
Voltage Clamp ◽  
Total Charge ◽  
Charge Movement ◽  
Cell Coupling ◽  
Β Cells ◽  
Patch Clamp Technique ◽  
Intact Mouse ◽  
Whole Cell ◽  
Junctional Conductance ◽  
Gap Junctional

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified β-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent β-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between β-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 β-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to −55 mV) gave rise to a slow capacitive current indicative of coupling between β-cells, but not in non-β-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial β-cell in an islet is in electrical contact with six to seven other β-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 K ATP channel conductance ( G K,ATP ) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC 50 of approximately 4 mM. Theoretical considerations indicate that the coupling between β-cells within the islet is sufficient to allow propagation of [Ca 2+ ] i waves to spread with a speed of approximately 80 μm s −1 , similar to that observed experimentally in confocal [Ca 2+ ] i imaging.

Quantitative analysis of dual whole-cell voltage-clamp determination of gap junctional conductance

1998 ◽  
Vol 436 (1) ◽  
pp. 141-151 ◽  
Author(s):  
H. V. M. Van Rijen ◽  
Ronald Wilders ◽  
Antoni C. G. Van Ginneken ◽  
Habo J. Jongsma
Keyword(s):  
Quantitative Analysis ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp ◽  
Junctional Conductance ◽  
Gap Junctional

Gap-junctional properties of electrically coupled skate horizontal cells in culture

1993 ◽  
Vol 10 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Haohua Qian ◽  
Robert Paul Malchow ◽  
Harris Ripps
Keyword(s):  
Receptive Field ◽  
Lucifer Yellow ◽  
Horizontal Cell ◽  
Electrical Coupling ◽  
Cell Voltage ◽  
Horizontal Cells ◽  
Electrical Synapse ◽  
Cell Coupling ◽  
Junctional Conductance ◽  
Gap Junctional

AbstractWhole-cell voltage-clamp recordings were used to examine the unusual pharmacological properties of the electrical coupling between rod-driven horizontal cells in skate retina as revealed previously by receptive-field measurements (Qian & Ripps, 1992). The junctional resistance was measured in electrically coupled cell pairs that had been enzymatically isolated and maintained in culture; the typical value was about 19.92 MΩ(n = 45), more than an order of magnitude lower than the nonjunctional membrane resistance. These data and the intercellular spread of the fluorescent dye Lucifer Yellow provide a good indication that skate horizontal cells are well coupled. The junctional conductance between cells was not modulated by the neurotransmitters dopamine (200 μM) or GABA (1 mM), nor was it affected by the membrane-permeable analogues of cAMP or cGMP, or the adenylate cyclase activator, forskolin. Although resistant to agents that have been reported to alter horizontal-cell coupling in cone-driven horizontal cells, the junctional conductance between paired horizontal cells of skate was greatly reduced by the application of 20 mM acetate, which is known to effectively reduce intracellular pH. Together with the results obtained in situ on the receptive-field properties of skate horizontal cells, these findings indicate that the gap-junctional properties of rod-driven horizontal cells of the skate are fundamentally different from those of cone-driven horizontal cells in other species. This raises the possibility that there is more than one class of electrical synapse on vertebrate horizontal cells.

Patch-clamp study reveals that the importance of connexin43-mediated gap junctional communication for ovarian folliculogenesis is strain specific in the mouse

2006 ◽  
Vol 290 (1) ◽  
pp. C290-C297 ◽  
Author(s):  
Dan Tong ◽  
Joanne E. I. Gittens ◽  
Gerald M. Kidder ◽  
Donglin Bai
Keyword(s):  
Patch Clamp ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Wild Type ◽  
Cell Coupling ◽  
Patch Clamp Technique ◽  
Capacitive Current ◽  
Genetic Ablation ◽  
Junctional Communication ◽  
Gap Junctional

Genetic ablation of connexin37 (Cx37) or connexin43 (Cx43), the two gap junction proteins expressed by mouse ovarian granulosa cells, has been shown to result in impaired follicle development. We used patch-clamp techniques to evaluate quantitatively the contribution of these connexins to gap junctional intercellular communication (GJIC) among granulosa cells. The coupling conductance derived from a voltage step-induced capacitive current transient was used as a measure of GJIC in cultured granulosa cells. Using this method, we determined that the conductance of wild-type (84.1 ± 28.6 nS; n = 6) and Cx37-deficient granulosa cells (83.7 ± 6.4 nS; n = 11) does not differ significantly ( P = 0.35), suggesting a limited contribution, if any, of Cx37 to granulosa cell coupling. In contrast, the conductance between granulosa cells of Cx43-deficient mice (2.6 ± 0.8 nS; n = 5) was not significantly different from that of single, isolated wild-type granulosa cells (2.5 ± 0.7 nS, n = 5; P = 0.83), indicating that Cx43-deficient granulosa cells were not electrically coupled. A direct measurement of transjunctional conductance between isolated granulosa cell pairs using a dual patch-clamp technique confirmed this conclusion. Interestingly, a partial rescue of folliculogenesis was observed when the Cx43-null mutation in C57BL/6 mice was crossed into the CD1 strain, and capacitive current measurement demonstrated that this rescue was not due to reestablishment of GJIC. These results demonstrate that folliculogenesis is impaired in the absence of GJIC between granulosa cells, but they also indicate that the severity is dependent on genetic background, a phenomenon that cannot be attributed to the expression of additional connexins.

Alpha2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells

1991 ◽  
Vol 11 (3) ◽  
pp. 147-157 ◽  
Author(s):  
Krister Bokvist ◽  
Carina Ämmälä ◽  
Per-Olof Berggren ◽  
Patrik Rorsman ◽  
Karin Wåhlander
Keyword(s):  
Adrenergic Stimulation ◽  
Β Cells ◽  
Adrenergic Agonist ◽  
Cell Interior ◽  
Pancreatic Β Cells ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Maximal Activation ◽  
Cell Recording

The effects of α2-adrenergic stimulation on the Ca2+-current in mouse pancreatic β-cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 μM) or the α2-adrenergic agonist clonidine (5 μM) failed to reduce the Ca2+-current, irrespective of whether intracellular GTP (or GTPγ S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca2+-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended to increase the Ca2+-current amplitude in the presence of the higher Ca2+-concentration. Ca2+-channel activation measured at 1.3 mM Ca2+ was not affected by clonidine. Half-maximal activation was observed at ≈−20 mV. In addition, when Ca2+-currents were recorded from intact β-cells, using the perforated patch whole-cell configuration, clonidine (1 μM) also failed to detectably affect the Ca2+-current. It is therefore suggested that the inhibition of β-cell electrical activity and insulin-secretion produced by α2-adrenoreceptor stimulation does not result from suppression of the L-type Ca2+-current.

scholarly journals Optical measurement of cell-to-cell coupling in intact heart using subthreshold electrical stimulation

2001 ◽  
Vol 281 (2) ◽  
pp. H533-H542 ◽  
Author(s):  
Fadi G. Akar ◽  
Bradley J. Roth ◽  
David S. Rosenbaum
Keyword(s):  
Fiber Orientation ◽  
Optical Measurement ◽  
Critical Role ◽  
Electrical Coupling ◽  
Membrane Properties ◽  
Cell Coupling ◽  
Close Proximity ◽  
Junctional Conductance ◽  
Gap Junctional ◽  
Intact Heart

Electrical coupling between myocytes plays a critical role in propagation, repolarization, and arrhythmias. On the basis of predictions from cable theory, we hypothesized that the cardiac space constant (λ) measured from the decay of subthreshold transmembrane potential (ST- V m) in space would provide an index of regional cell-to-cell coupling in the intact heart. With the use of voltage-sensitive dyes, the distribution of ST- V m was measured from hundreds of sites in close proximity to the site of subthreshold stimulation. λ was calculated from the exponential decay of ST- V min space. Consistent with known directional differences in axial resistance, the spatial distribution of ST- V mwas strongly dependent on fiber orientation, because λ was significantly ( P < 0.001) longer along (1.5 ± 0.1 mm) compared with across (0.8 ± 0.1 mm) fibers. There was a close linear relationship ( P < 0.001) between conduction velocity (CV) and λ along all fiber angles tested. Reducing gap junctional conductance by heptanol reversibly decreased CV and λ in parallel by ∼50%. In contrast, sodium channel blockade by flecainide slowed CV by 40% but had no effect on λ, reaffirming that λ was an index of passive but not active membrane properties. These data establish the feasibility of measuring λ as an index of cell-to-cell coupling in the intact heart, and indicate strong dependency of λ on fiber orientation and pharmacological alterations of gap junction conductance.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2020 ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the characterization of channel properties or drug actions, such limits being different from one channel to another. In the present study, we use kinetic models of a voltage-gated sodium channel and a voltage-gated potassium channel to edict simple guidelines for good practice of whole-cell voltage-clamp recordings.

scholarly journals Characterization of the calcium-sensitive voltage-gated delayed rectifier potassium channel in isolated guinea pig hepatocytes.

1994 ◽  
Vol 104 (1) ◽  
pp. 147-171 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Horikawa ◽  
T Aramaki ◽  
H Okumura
Keyword(s):  
Guinea Pig ◽  
Voltage Clamp ◽  
Exponential Function ◽  
Delayed Rectifier ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Voltage Gated ◽  
Tail Current ◽  
Inside Out

The voltage-dependent K+ channel was examined in enzymatically isolated guinea pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. The resting membrane potential in isolated hepatocytes was -25.3 +/- 4.9 mV (n = 40). Under the whole-cell voltage-clamp, the time-dependent delayed rectifier outward current was observed at membrane potentials positive to -20 mV at physiological temperature (37 degrees C). The reversal potential of the current, as determined from tail current measurements, shifted by approximately 57 mV per 10-fold change in the external K+ concentration. In addition, the current did not appear when K+ was replaced with Cs+ in the internal and external solutions, indicating that the current was carried by K+ ions. The envelope test of the tails demonstrated that the growth of the tail current followed that of the current activation. The ratio between the activated current and the tail amplitude was constant during the depolarizing step. The time course of growth and deactivation of the tail current were best described by a double exponential function. The current was suppressed in Ca(2+)-free, 5 mM EGTA internal or external solution (pCa &gt; 9). The activation curve (P infinity curve) was not shifted by changing the internal Ca2+ concentration ([Ca2+]i). The current was inhibited by bath application of 4-aminopyridine or apamin. alpha 1-Adrenergic stimulation with noradrenaline enhanced the current but beta-adrenergic stimulation with isoproterenol had no effect on the current. In single-channel recordings from outside-out patches, unitary current activity was observed by depolarizing voltage-clamp steps whose slope conductance was 9.5 +/- 2.2 pS (n = 10). The open time distribution was best described by a single exponential function with the mean open lifetime of 18.5 +/- 2.6 ms (n = 14), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 2.0 +/- 0.3 ms (n = 14) and that for the slow component of 47.7 +/- 5.9 ms (n = 14). Ensemble averaged current exhibited delayed rectifier nature which was consistent with whole-cell measurements. In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The concentration of Ca2+ at the half-maximal activation was 0.031 microM. These results suggest that guinea pig hepatocytes possess voltage-gated delayed rectifier K+ channels which are modified by intracellular Ca2+.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

AbstractThe patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

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