RESUMO

In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.

Assuntos

RESUMO

Reconciling protein functional data with crystal structure is arduous because rare conformations or crystallization artifacts occur. Here we present a tool to validate the dimensions of open pore structures of potassium-selective ion channels. We used freely available algorithms to calculate the molecular contour of the pore to determine the effective internal pore radius (r(E)) in several K-channel crystal structures. r(E) was operationally defined as the radius of the biggest sphere able to enter the pore from the cytosolic side. We obtained consistent r(E) estimates for MthK and Kv1.2/2.1 structures, with r(E) = 5.3-5.9 Å and r(E) = 4.5-5.2 Å, respectively. We compared these structural estimates with functional assessments of the internal mouth radii of capture (r(C)) for two electrophysiological counterparts, the large conductance calcium activated K-channel (r(C) = 2.2 Å) and the Shaker Kv-channel (r(C) = 0.8 Å), for MthK and Kv1.2/2.1 structures, respectively. Calculating the difference between r(E) and r(C), produced consistent size radii of 3.1-3.7 Å and 3.6-4.4 Å for hydrated K(+) ions. These hydrated K(+) estimates harmonize with others obtained with diverse experimental and theoretical methods. Thus, these findings validate MthK and the Kv1.2/2.1 structures as templates for open BK and Kv-channels, respectively.

RESUMO

Mammals maintain homeostatic control of their body temperature. Therefore, these organisms are expected to have adaptations that confer the ability to detect and react to both self and ambient temperature. Temperature-activated ion channels have been discovered to be the primary molecular determinants of thermosensation. The most representative group of these determinants constitutes members of the transient receptor potential superfamily, TRP, which are activated by either low or high temperatures covering the whole range of physiologically relevant temperatures. This review makes a critical assessment of existing analytical methods of temperature-activated TRP channel mechanisms using the cold-activated TRPM8 channel as a paradigm.

RESUMO

The transient receptor potential ion channel TRPA1 confers the ability to detect tissue damaging chemicals to sensory neurons and as a result mediates chemical nociception in vivo. Mouse TRPA1 is activated by electrophilic compounds such as mustard-oil and several physical stimuli such as cold temperature. Due to its sensory function inhibition of TRPA1 activity might provide an effective treatment against chronic and inflammatory pain. Therefore, TRPA1 has become a target for the development of analgesic drugs. 6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (Compound 31) has been identified by a chemical screen and lead optimization as an inhibitor of chemical activation of TRPA1. However, the structures or domains of TRPA1 that mediate the inhibitory effect of Compound 31 are unknown. Here, we screened 12,000 random mutant clones of mouse TRPA1 for their sensitivity to mustard-oil and the ability of Compound 31 to inhibit chemical activation by mustard-oil. In addition, we separately screened this mutant library while stimulating it with cold temperatures. We found that the single-point mutation I624N in the N-terminus of TRPA1 specifically affects the sensitivity to mustard-oil, but not to cold temperatures. This is evidence that sensitivity of TRPA1 to chemicals and cold temperatures is conveyed by separable mechanisms. We also identified five mutations located within the pore domain that cause loss of inhibition by Compound 31. This result demonstrates that the pore-domain is a regulator of chemical activation and suggests that Compound 31 might be acting directly on the pore-domain.

Assuntos

RESUMO

Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na(+) overloads elicited by engagement of the Na(+)-glutamate cotransporter with d-aspartate or application of the Na(+) ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K(+) stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K(+) ended immediately but the stimulation by glutamate persisted unabated for >20 min. Both stimulations required an active Na(+)/K(+) ATPase pump. By showing that small rises in extracellular K(+) mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.

Assuntos

RESUMO

In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6-ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.

Assuntos