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Resting potentials (Vm) were measured in mouse Leydig cells, using the whole-cell patch-clamp technique. In contrast to conventional microelectrode measurements, where a biphasic potential was observed, we recorded a stable Vm around -32.2 ± 1.2 mV (mean ± SEM, n = 159), at 25°C, and an input resistance larger than 2.7 × 109 W. Although Vm is sensitive to changes in the extracellular concentrations of potassium and chloride, the relationship between Vm and these ions' concentrations cannot be described by either the Goldman-Hodgkin-Katz or the Nernst equation. Perifusing cells with potassium-free solution or 10?3 M ouabain induced a marked depolarization averaging 20.1 ± 3.2 mV (n = 9) and 23.1 ± 2.8 mV, (n = 7), respectively. Removal of potassium or addition of ouabain with the cell voltage-clamped at its Vm, resulted in an inwardly directed current, due to inhibition of the Na+K+ATPase. The pump current increased with temperature with a Q10 coefficient of 2.3 and had an average value of -6.5 ± 0.4 pA (n = 21) at 25°C. Vm also varied strongly with temperature, reaching values as low as -9.2 ± 1.2 mV (n = 22) at 15°C. Taking the pump current at 25°C and a minimum estimate for the membrane input resistance, we can see that the Na+K+ATPase could directly contribute with 17.7 mV to the Vm of Leydig cells, which is a major fraction of the ?32.2 ± 1.2 mV (n = 159) observed. [ABSTRACT FROM AUTHOR]

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B-microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

The major neuronal gap junction protein connexin36 (Cx36) exhibits the remarkable property of "run-up", in which junctional conductance typically increases by 10-fold or more within 5-10min following cell break-in with patch pipettes. Such conductance "run-up" is a unique property of Cx36, as it has not been seen in cell pairs expressing other connexins. Because of the recent observation describing CaMKII binding and phosphorylation sites in Cx36 and evidence that calmodulin dependent protein kinase II (CaMKII) may potentiate electrical coupling in neurons of teleosts, we have explored whether CaMKII activates mammalian Cx36. Consistent with this hypothesis, certain Cx36 mutants lacking the CaMKII binding and phosphorylation sites or wild type Cx36 treated with certain cognate peptides corresponding to binding or phosphorylation sites blocked or strongly attenuated run-up of junctional conductance. Likewise, KN-93, an inhibitor of CaMKII, blocked run-up, as did a membrane permeable peptide corresponding to the CaMKII autoinhibitory domain. Furthermore, run-up was blocked by phosphatase delivered within the pipette and not affected by treatment with the phosphatase inhibitor okadaic acid. These results imply that phosphorylation by CaMKII strengthens junctional currents of Cx36 channels, thereby conferring functional plasticity on electrical synapses formed of this protein., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Voltage-gated K(+) (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K(+) channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.

2-Aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor modulator, inhibits capacitive current transients measured in normal rat kidney and human embryonic kidney 293 cells, an indication of blocking gap junction channels between these cells. Here, we used the dual whole-cell patch-clamp method to study the actions of 2-APB on gap junction channels formed by selected connexins expressed in a communication-deficient neuroblastoma cell line (N2A). 2-APB dose-dependently and reversibly blocked junctional currents of connexin (Cx) 50 gap junction channels. The concentration-inhibition curve of 2-APB on the junctional current indicated an IC(50) of 3.7 microM, lower than that of most gap junction inhibitors. At a concentration of 20 microM, 2-APB also significantly blocked junctional conductance in cell pairs coupled by Cx26, Cx30, Cx36, Cx40, and Cx45 but did not appreciably affect coupling in cell pairs expressing Cx32, Cx43, and Cx46. Although concentration inhibition curves of 2-APB on Cx36 channels were similar to Cx50 (Cx36; IC(50), 3.0 microM), IC(50) values were higher for Cx43 (51.6 microM), Cx45 (18.1 microM), and Cx46 (29.4 microM). The blocking action of 2-APB did not substantially alter transjunctional voltage-dependent gating of Cx50 gap junction channels, and recordings from poorly coupled pairs of Cx50-transfected N2A cells indicated that 2-APB reduced gap junction channel open probability without changing the main state single-channel conductance. The differential efficacy of block by 2-APB of gap junction channels formed by different connexins may provide a useful tool that could be exploited in gap junction research to selectively block certain gap junction channel subtypes.

Pathophysiological changes in arterial smooth muscle structure and function occur with aging and there are a number of reports illustrating reductions in vascular responsiveness with aging. While much is known about arterial remodeling and functional adaptations with aging, very little is known about the biophysical adaptations in individual arterial myocytes. Cytosolic Ca2+ signaling, involving activation of L-type Ca2+ channels on the plasma membrane as well as InsP3 and ryanodine receptors on the sarcoplasmic reticulum, is integral to vascular tone and reactivity. Thus, we tested the hypothesis that aging results in reductions in the functional expression of L-type channels and temporal aspects of ryanodine receptor and InsP3 receptor Ca2+ signaling, in mesenteric arterial smooth muscle cells isolated from 6 and 30 months old C57Bl/6 mice. Comparisons of L-type current activity were made using dialyzed, whole-cell voltage-clamp techniques and Ba2+ as charge carrier. Ca2+ signaling was measured using fura-2 fluorescence microscopy techniques. Cell morphological changes were also investigated using electrophysiological and immunocytochemical approaches. The amplitudes of L-type Ca2+ currents were increased in older mice, but this was associated with membrane surface area increases of approximately 50%, due to increases in cell length not cell width. Consequently, L-type Ca2+ current densities were preserved with age, indicating functional channel expression was unchanged. In contrast, aging was associated with decrements in Ca2+ signaling in response to either ryanodine receptor stimulation by caffeine or InsP3 receptor activation with phenylephrine. These changes with aging may be related to the previously reported depression in myogenic reactivity.

Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels.

Autoantibodies against muscarinic and adrenergic receptors have been found in the sera of patients with idiopathic dilated cardiomyopathy (IDC) and Chagas disease, but it is still unclear whether they can functionally interact with their respective receptors to modulate cardiac functions. In this study, our goal was to detect the presence of those antibodies in the sera of patients with IDC and characterize their electrophysiological effects on cardiomyocytes from rabbits. By using ELISA immunoassays, we detected high titers of antibodies against muscarinic M2 receptors in the sera of all IDC patients, whereas the detection of antibodies against the beta1-receptor occurred in 50% of them. Electrophysiological experiments using the whole cell configuration of the patch-clamp technique showed that sera from 43% of IDC patients induced a significant decrease (approximately 26%) in isoproterenol-stimulated L-type Ca2+ currents in rabbit ventricular myocytes, whereas the sera from healthy blood donors failed to do so. As expected, IDC sera also decreased the action potential duration (by 10.5%) due to a shortening of the plateau phase. Sera that reduced isoproterenol-stimulated L-type Ca2+ currents did not cause any effect on K+ currents. We conclude that sera from IDC patients have autoantibodies, which interact with muscarinic M2 receptors of rabbit cardiomyocytes, acting in an agonist-like fashion. This action results in changes in electrogenesis, which, as often observed in patients with IDC, could initiate ventricular arrhythmias that lead to sudden death.