It has been proposed that ephaptic interactions in intercalated discs, mediated by extracellular potentials, contribute to cardiac impulse propagation when gap junctional coupling is reduced. However, experiments demonstrating ephaptic effects on the cardiac Na current (I ) are scarce. Furthermore, Na channels form clusters around gap junction plaques, but the electrophysiological significance of these clusters has never been investigated. In patch clamp experiments with HEK cells stably expressing human Na 1.5 channels, we examined how restricting the extracellular space modulates I elicited by an activation protocol. In parallel, we developed a high-resolution computer model of the intercalated disc to investigate how the distribution of Na channels influences ephaptic interactions. Approaching the HEK cells to a non-conducting obstacle always increased peak I at step potentials near the threshold of I activation and decreased peak I at step potentials far above threshold (7 cells, P = 0.0156, Wilcoxon signed rank test). These effects were consistent with corresponding control simulations with a uniform Na channel distribution. In the intercalated disc computer model, redistributing the Na channels into a central cluster of the disc potentiated ephaptic effects. Moreover, ephaptic impulse transmission from one cell to another was facilitated by clusters of Na channels facing each other across the intercellular cleft when gap junctional coupling was reduced. In conclusion, our proof-of-principle experiments demonstrate that confining the extracellular space modulates cardiac I , and our simulations reveal the functional role of the aggregation of Na channels in the perinexus. These findings highlight novel concepts in the physiology of cardiac excitation.

Acid-sensing ion channels (ASICs) are H +-activated neuronal Na + channels. They are involved in fear behavior, learning, neurodegeneration after ischemic stroke and in pain sensation. ASIC activation has so far been studied only with fast pH changes, although the pH changes associated with many roles of ASICs are slow. It is currently not known whether slow pH changes can open ASICs at all. Here, we investigated to which extent slow pH changes can activate ASIC1a channels and induce action potential signaling. To this end, ASIC1a current amplitudes and charge transport in transfected Chinese hamster ovary cells, and ASIC-mediated action potential signaling in cultured cortical neurons were measured in response to defined pH ramps of 1-40 s duration from pH 7.4 to pH 6.6 or 6.0. A kinetic model of the ASIC1a current was developed and integrated into the Hodgkin-Huxley action potential model. Interestingly, whereas the ASIC1a current amplitude decreased with slower pH ramps, action potential firing was higher upon intermediate than fast acidification in cortical neurons. Indeed, fast pH changes (<4 s) induced short action potential bursts, while pH changes of intermediate speed (4-10 s) induced longer bursts. Slower pH changes (>10 s) did in many experiments not generate action potentials. Computer simulations corroborated these observations. We provide here the first description of ASIC function in response to defined slow pH changes. Our study shows that ASIC1a currents, and neuronal activity induced by ASIC1a currents, strongly depend on the speed of pH changes. Importantly, with pH changes that take >10 s to complete, ASIC1a activation is inefficient. Therefore, it is likely that currently unknown modulatory mechanisms allow ASIC activity in situations such as ischemia and inflammation.

Background: Na v 1.5 cardiac Na + channel mutations can cause arrhythmogenic syndromes. Some of these mutations exert a dominant negative effect on wild-type channels. Recent studies showed that Na + channels can dimerize, allowing coupled gating. This leads to the hypothesis that allosteric interactions between Na + channels modulate their function and that these interactions may contribute to the negative dominance of certain mutations. Methods: To investigate how allosteric interactions affect microscopic and macroscopic channel function, we developed a modeling paradigm in which Markovian models of two channels are combined. Allosteric interactions are incorporated by modifying the free energies of the composite states and/or barriers between states. Results: Simulations using two generic 2-state models (C-O, closed-open) revealed that increasing the free energy of the composite states CO/OC leads to coupled gating. Simulations using two 3-state models (closed-open-inactivated) revealed that coupled closings must also involve interactions between further composite states. Using two 6-state cardiac Na + channel models, we replicated previous experimental results mainly by increasing the energies of the CO/OC states and lowering the energy barriers between the CO/OC and the CO/OO states. The channel model was then modified to simulate a negative dominant mutation (Na v 1.5 p.L325R). Simulations of homodimers and heterodimers in the presence and absence of interactions showed that the interactions with the variant channel impair the opening of the wild-type channel and thus contribute to negative dominance.

Background: The ability to differentiate patient-specific human induced pluripotent stem cells (hiPSC) into cardiac myocytes (hiPSC-CM) offers novel perspectives for cardiovascular research. A number of studies, which reported mainly on current-voltage curves used hiPSC-CM to model voltage-gated Na þ channel (Na v ) dysfunction. However, the expression patterns and precise biophysical and pharmacological properties of Na v channels from hiPSC-CM remain unknown. Our objective was to study the characteristics of Na v channels from hiPSC-CM and assess the appropriateness of this novel cell model. Methods: We generated hiPSC-CM using the recently described monolayer-based differentiation protocol. Results: hiPSC-CM expressed cardiac-specific markers, exhibited spontaneous electrical and contractile activities, and expressed distinct Na v channels subtypes. Electrophysiological, pharmacological, and molecular characterizations revealed that, in addition to the main Na v 1.5 channel, the neuronal TTX-sensitive Na v 1.7 channel was also significantly expressed in hiPSC-CM. Most of the Na þ currents were resistant to tetrodotoxin block. Therapeutic concentrations of lidocaine, a class I antiarrhythmic drug, also inhibited Na þ currents in a use-dependent manner. Na v 1.5 and Na v 1.7 expression and maturation patterns of hiPSC-CM and native Human cardiac tissues appeared to be similar. The four Na v b regulatory subunits were expressed in hiPSC-CM, with b3 being the preponderant subtype. Conclusions: The findings indicated that hiPSC-CM robustly express Na v 1.5 channels which exhibited molecular and pharmacological properties similar to those in native cardiac tissues. Interestingly, neuronal Na v 1.7 channels were also expressed in hiPSC-CM and are expected to be responsible for the TTX-sensitive Na v current.