Although rat brain Nav1.3 voltage-gated sodium channels have been expressed and studied in Xenopus oocytes, these channels have not been studied after their expression in mammalian cells. We characterized the properties of the rat brain Nav1.3 sodium channels expressed in human embryonic kidney (HEK) 293 cells. Nav1.3 channels generated fast-activating and fastinactivating currents. Recovery from inactivation was relatively rapid at negative potentials (ϽϪ80 mV) but was slow at more positive potentials. Development of closed-state inactivation was slow, and, as predicted on this basis, Nav1.3 channels generated large ramp currents in response to slow depolarizations. Coexpression of ␤3 subunits had small but significant effects on the kinetic and voltage-dependent properties of Nav1.3 currents in HEK 293 cells, but coexpression of ␤1 and ␤2 subunits had little or no effect on Nav1.3 properties. Nav1.3 channels, mutated to be tetrodotoxin-resistant (TTX-R), were expressed in SNS-null dorsal root ganglion (DRG) neurons via biolistics and were compared with the same construct expressed in HEK 293 cells. The voltage dependence of steadystate inactivation was ϳ7 mV more depolarized in SNS-null DRG neurons, demonstrating the importance of background cell type in determining physiological properties. Moreover, consistent with the idea that cellular factors can modulate the properties of Nav1.3, the repriming kinetics were twofold faster in the neurons than in the HEK 293 cells. The rapid repriming of Nav1.3 suggests that it contributes to the acceleration of repriming of TTX-sensitive (TTX-S) sodium currents that are seen after peripheral axotomy of DRG neurons. The relatively rapid recovery from inactivation and the slow closed-state inactivation kinetics of Nav1.3 channels suggest that neurons expressing Nav1.3 may exhibit a reduced threshold and/or a relatively high frequency of firing.

Tetrodotoxin-resistant sodium currents contribute to the somal and axonal sodium currents of small diameter primary sensory neurons, many of which are nociceptive. NaN is a recently described tetrodotoxin-resistant sodium channel expressed preferentially in IB4-labeled dorsal root ganglion (DRG) neurons. We employed an antibody raised to a NaN specific peptide to show that NaN is preferentially localized along axons of IB4-positive unmyelinated fibers in the sciatic nerve and in axon terminals in the cornea. NaN immunoreactivity was also found at some nodes of Ranvier of thinly myelinated axons of the sciatic nerve, where it was juxtaposed to Kv1.2 potassium channel immunoreactivity. This distribution of NaN is consistent with a role for NaN sodium channels in nociceptive transmission.

Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCI) to medium containing lower levels of K+ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad‐spectrum caspase inhibitors, tert‐butoxycarbonyl‐Asp·(O‐methyl)·fluoromethyl ketone and benzyloxycarbonyl‐Val‐Ala‐Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N‐acetyl Tyr‐Val‐Ala‐Asp·chloromethyl ketone (YVAD·cmk), an interleukin‐1β converting enzyme‐preferring inhibitor, and N‐acetyl Asp‐Glu‐Val‐Asp·fluoromethyl ketone (DEVD·fmk), a CPP32‐preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin‐1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP‐ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD‐sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+‐deprived granule neurons.

No abstract

A 30-year-old male presented with severe, warm autoimmune hemolysis 17 months subsequent to a matched, unrelated peripheral hematopoietic stem cell transplant. The patient responded poorly to conventional therapy with steroids and immunoglobulin, prompting the initiation of rituximab. On account of persistent, severe hemolysis, therapeutic plasma exchange was employed as a bridge until the rituximab therapy became effective. Immediately following plasmapheresis, the patient demonstrated clinical improvement followed by attenuation of the hemolysis and improved reticulocytosis. The hemoglobin concentration and reticulocyte index demonstrated further improvement following subsequent doses of rituximab and continued following the cessation of plasmapheresis. This case suggests the utility of plasmapheresis and rituximab in severe, life-threatening cases of warm autoimmune hemolytic anemia refractory to conventional therapy.