The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

We evaluated the contributions of somatic and dendritic impulses to the receptive field dimensions of amacrine cells in the amphibian retina. For this analysis, we used the NEURON simulation program with a multicompartmental, multichannel model of an On-Off amacrine cell with a three-dimensional structure obtained through computer tracing techniques. Simulated synaptic inputs were evenly spaced along the dendritic branches and organized into eight annuli of increasing radius. The first set of simulations activated each ring progressively to simulate an area summation experiment, while a second approach activated each annulus individually. Both sets of simulations were done with and without the presence of Na channels in the dendrites and soma. Unexpectedly, the receptive field dimensions observed in the area summation simulations was often smaller than that predicted from the summation of the annular simulations. Collisions of action potentials moving in opposite directions in the dendrites largely accounted for this contraction in receptive field size for the area summation studies. The presence of dendritic Na channels increased the size of the receptive field beyond that achieved in their absence and allowed the physiological size of the receptive field to approximate the physical dimensions of the dendritic tree. This receptive field augmentation was the result of impulse generating ability in the dendrites which enhanced the signal observed at the soma. These simulations provide a plausible mechanistic explanation for physiological recordings from amacrine cells that show similar phenomena.

Background and objective: Similar doses of ketamine are employed in regional and general anaesthesia. In contrast to commonly used local anaesthetic agents, accidental systemic application of local anaesthetic doses of ketamine will not result in seizure, dysrhythmia or cardiovascular depression. However, there is some doubt about the quality of regional analgesia induced by ketamine. As human sodium channels constitute an important molecular target of local anaesthetics, the study was designed to establish concentration-dependent effects of ketamine on conductance, steady-state activation and steady-state inactivation of human neuronal sodium channels. This information — that has, so far, not been published — will help to characterize further local anaesthetic properties of ketamine.

Methods: Whole-cell patch-clamp recordings were made of sodium channels natively expressed in human neuroblastoma SH-SY5Y cells.

Results: The sodium channels activated at a threshold of −60 mV and exhibited a maximal peak current at −10 mV. The voltage of half-maximal activation was −20 mV. The Na+ currents depended on the prepulse potential. The voltage of half-maximal inactivation was —80 mV. Ketamine inhibited the sodium conductance in a concentration-dependent manner (IC50 = 1140 µmol). A concentration-dependent hyperpolarizing shift of both steady-state activation and steady-state inactivation accounted for at most 5 mV.

Conclusions: The effects of ketamine on these human ion channels occur at clinical concentrations. They are consistent with the local anaesthetic action of ketamine. Whether ketamine helps to decrease the incidence of severe side-effects during regional anaesthesia needs to be addressed in further clinical studies.

Background and Objective To investigate the response to general anaesthetics of different sodium channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human ventricular muscle and compared them with existing data from human brain channels.

Methods Sodium channels from preparations of human ventricular muscle were incorporated into planar lipid bilayers in the presence of batrachotoxin, a sodium channel activator. Single channel currents were recorded in symmetrical 100mmolL−1 and 500mmolL−1 NaCl before and after the addition of the anaesthetic pentobarbital (0.34–1.34mmolL−1).

Results The blocking effect of pentobarbital on the fractional open time had an IC50 of 690 μmol L−1 in 500 mmolL−1 NaCl, whereas it had a significantly lower IC50 of 400 μmol L−1 in 100 mmolL−1 NaCl. Pentobarbital caused a significant shift of steady-state activation to hyperpolarized potentials (fmax = −42 mV, IC50=2 mmolL−1). This effect was independent of NaCl concentration.

Conclusion Despite pharmacological and electrophysiological differences between human cardiac and human brain sodium channels their responses to pentobarbital are similar. The finding of channel block being dependent on the electrolyte concentration is novel for sodium channels.