N terminus is key to the dominant negative suppression of CaV2 calcium channels: implications for episodic ataxia type 2

Expression of the calcium channels CaV2.1 and CaV2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in CaV2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both CaV2.1 and CaV2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg52 and Arg54) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of CaV2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in CaV2.2 protein level, and this is also prevented by mutation of Arg52 and Arg54 to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg52, Arg54 motif are involved in the processes underlying dominant negative suppression

Dominant-negative calcium channel suppression by truncated constructs involves a kinase implicated in the unfolded protein response

Expression of the calcium channel Ca(V)2.2 is markedly suppressed by coexpression with truncated constructs of Ca(V)2.2. Furthermore, a two-domain construct of Ca(V)2.1 mimicking an episodic ataxia-2 mutation strongly inhibited Ca(V)2.1 currents. We have now determined the specificity of this effect, identified a potential mechanism, and have shown that such constructs also inhibit endogenous calcium currents when transfected into neuronal cell lines. Suppression of calcium channel expression requires interaction between truncated and full-length channels, because there is inter-subfamily specificity. Although there is marked cross-suppression within the Ca(V)2 calcium channel family, there is no cross-suppression between Ca(V)2 and Ca(V)3 channels. The mechanism involves activation of a component of the unfolded protein response, the endoplasmic reticulum resident RNA-dependent kinase (PERK), because it is inhibited by expression of dominant-negative constructs of this kinase. Activation of PERK has been shown previously to cause translational arrest, which has the potential to result in a generalized effect on protein synthesis. In agreement with this, coexpression of the truncated domain I of Ca(V)2.2, together with full-length Ca(V)2.2, reduced the level not only of Ca(V)2.2 protein but also the coexpressed alpha2delta-2. Thapsigargin, which globally activates the unfolded protein response, very markedly suppressed Ca(V)2.2 currents and also reduced the expression level of both Ca(V)2.2 and alpha2delta-2 protein. We propose that voltage-gated calcium channels represent a class of difficult-to-fold transmembrane proteins, in this case misfolding is induced by interaction with a truncated cognate Ca(V) channel. This may represent a mechanism of pathology in episodic ataxia-2

Indirect actions of bradykinin on neonatal rat dorsal root ganglion neurones: a role for non-neuronal cells as nociceptors

In this study we have investigated the action of bradykinin (Bk) on cultured neonatal rat dorsal root ganglion (DRG) cells, with the aim of elucidating whether the neuronal response to Bk is influenced by association with non-neuronal satellite cells.Bradykinin (100 nm) evoked an inward current (IBk) in 51 of 58 voltage clamped DRG neurones (holding potential (Vh) =−80 mV) that were in contact with non-neuronal satellite cells.Bradykinin failed to evoke an inward current in isolated DRG neurones (Vh=−80 mV) that were not in contact with non-neuronal satellite cells (n = 41).The lack of neuronal response to Bk was not influenced by time in culture. Bradykinin failed to evoke a response in isolated neurones through 1–5 days in culture. By contrast neurones in contact with satellite cells responded to Bk throughout the same time period.Failure of isolated neurones to respond to Bk was not due to the replating procedure or to selective subcellular distribution of receptors/ion channels to the processes rather than the somata of neurones.Using Indo-1 AM microfluorimetry Bk (100 nm) was demonstrated to evoke an intracellular Ca2+ increase (CaBk) in DRG neurones in contact with non-neuronal satellite cells and in isolated neurones.These data suggest that the inward current response to Bk requires contact between DRG neurones and non-neuronal satellite cells. This implies an indirect mechanism of action for Bk via the non-neuronal cells, which may perform a nociceptive role. However, Bk can also act directly on the neurones, since it evokes CaBk in isolated neurones. The relationship between CaBk and the Bk-induced inward current is unknown at present

Klinische Sportmedizin

Background: The mandatory medical fitness examinations of firefighters and occupational divers with ergometry are important, but in Germany they are based on two criticized models, Reiterers model and the PWC-model.Aim: We examine the weaknesses of these models and offer alternatives. Besides, we discuss other problems of the fitness examinations.Methods: In bicycle ergometry tests with 8583 firefighters we collected data concerning age, mass and final achievements and developed mathematical models that allow us to estimate the Median of the final achievement from age and mass.The special relevance here is that our models are based on data of firefighters and are therefore better suited as basis of medical fitness tests in contrast to Reiterers model which was conceived for another purpose.Results: Reiterers model is based on measurements with less-fit test persons. As expected, on average it underestimates the achievements. The structure is clearly not as precise as our models. The PWC model is even less precise and is inclined to overestimate the achievements. Our algorithm Pincremental well describes the physical fitness of firefighters and can be used to construct reference models, based on gender, form of ergometry and failure rate.Conclusion: We recommend the use of new models for medical fitness examinations. When using a bicycle ergometer, a gradual increase protocol with steeper gradient instead of the present increment should come into use. The fitness examinations of firefighters should be based more on data from the job and contain job-specific forms of ergometry, at least in part.KEY WORDS: Bicycle Ergometry, Firefighters, Fitness Tests, Cardiopulmonary Performanc

Bradykinin evokes a Ca2+-activated chloride current in non-neuronal cells isolated from neonatal rat dorsal root ganglia

We have studied the effect of bradykinin (Bk) on fibroblast-like satellite (FLS) cells isolated from cultures of neonatal rat dorsal root ganglia (DRG).In voltage-clamped FLS cells Bk evoked an inward current response that was concentration dependent with a half-maximal concentration of 2 nM.In indo-1 AM-loaded FLS cells Bk evoked a rise in intracellular Ca2+ that was concentration dependent with a half-maximal concentration of 1 nM.The FLS cells still produced an inward current in response to Bk in the absence of extracellular Ca2+ but the response was inhibited if the intracellular concentration of EGTA was increased from 0.5 to 5 mM, which suggests that the inward current was dependent on the release and subsequent rise of intracellular Ca2+.The reversal potential of the Bk-induced inward current was consistent with the current being due to an increase in Cl− conductance and shifted in a Nernstian manner when the intracellular Cl− concentration was reduced.The inward current response to Bk was blocked by the B2 receptor antagonist HOE-140, which indicates that the response was due to activation of B2 receptors.The data suggest that Bk evokes a rise in intracellular Ca2+ and activation of a Ca2+-activated Cl− conductance in the FLS cells and raise the possibility that FLS cells contribute to the pro-inflammatory effects of Bk in DRG