Electrostatic Contributions of Aromatic Residues in the Local Anesthetic Receptor of Voltage-Gated Sodium Channels

Antiarrhythmics, anticonvulsants, and local anesthetics target voltage-gated sodium channels, decreasing excitability of nerve and muscle cells. Channel inhibition by members of this family of cationic, hydrophobic drugs relies on the presence of highly conserved aromatic residues in the pore-lining S6 segment of the fourth homologous domain of the channel. We tested whether channel inhibition was facilitated by an electrostatic attraction between lidocaine and {pi} electrons of the aromatic rings of these residues, namely a cation-{pi} interaction. To this end, we used the in vivo nonsense suppression method to incorporate a series of unnatural phenylalanine derivatives designed to systematically reduce the negative electrostatic potential on the face of the aromatic ring. In contrast to standard point mutations at the same sites, these subtly altered amino acids preserve the wild-type voltage dependence of channel activation and inactivation. Although these phenylalanine derivatives have no effect on low-affinity tonic inhibition by lidocaine or its permanently charged derivative QX-314 at any of the substituted sites, high-affinity use-dependent inhibition displays substantial cation-{pi} energetics for 1 residue only: Phe1579 in rNaV1.4. Replacement of the aromatic ring of Phe1579 by cyclohexane, for example, strongly reduces use-dependent inhibition and speeds recovery of lidocaine-engaged channels. Channel block by the neutral local anesthetic benzocaine is unaffected by the distribution of {pi} electrons at Phe1579, indicating that our aromatic manipulations expose electrostatic contributions to channel inhibition. These results fine tune our understanding of local anesthetic inhibition of voltage-gated sodium channels and will help the design of safer and more salutary therapeutic agents

A Cation–π Interaction between Extracellular TEA and an Aromatic Residue in Potassium Channels

Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation–{pi} interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation–{pi} binding ability of an aromatic ring, progressively increased the inhibitory constant Ki for the TEA block of Shaker. A larger increase in Ki was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation–{pi} component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation–{pi} mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation–{pi} interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy

A Cation-π Interaction Discriminates among Sodium Channels That Are Either Sensitive or Resistant to Tetrodotoxin Block

Voltage-gated sodium channels control the upstroke of the action potential in excitable cells of nerve and muscle tissue, making them ideal targets for exogenous toxins that aim to squelch electrical excitability. One such toxin, tetrodotoxin (TTX), blocks sodium channels with nanomolar affinity only when an aromatic Phe or Tyr residue is present at a specific location in the external vestibule of the ion-conducting pore. To test whether TTX is attracted to Tyr401 of NaV1.4 through a cation-{pi} interaction, this aromatic residue was replaced with fluorinated derivatives of Phe using in vivo nonsense suppression. Consistent with a cation-{pi} interaction, increased fluorination of Phe401, which reduces the negative electrostatic potential on the aromatic face, caused a monotonic increase in the inhibitory constant for block. Trifluorination of the aromatic ring decreased TTX affinity by ~50-fold, a reduction similar to that caused by replacement with the comparably hydrophobic residue Leu. Furthermore, we show that an energetically equivalent cation-{pi} interaction underlies both use-dependent and tonic block by TTX. Our results are supported by high level ab initio quantum mechanical calculations applied to a model of TTX binding to benzene. Our analysis suggests that the aromatic side chain faces the permeation pathway where it orients TTX optimally and interacts with permeant ions. These results are the first of their kind to show the incorporation of unnatural amino acids into a voltage-gated sodium channel and demonstrate that a cation-{pi} interaction is responsible for the obligate nature of an aromatic at this position in TTX-sensitive sodium channels

An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels

Slow inactivation of Kv1 channels involves conformational changes near the selectivity filter. We examine such changes in Shaker channels lacking fast inactivation by considering the consequences of mutating two residues, T449 just external to the selectivity filter and V438 in the pore helix near the bottom of the selectivity filter. Single mutant T449F channels with the native V438 inactivate very slowly, and the canonical foot-in-the-door effect of extracellular tetraethylammonium (TEA) is not only absent, but the time course of slow inactivation is accelerated by TEA. The V438A mutation dramatically speeds inactivation in T449F channels, and TEA slows inactivation exactly as predicted by the foot-in-the-door model. We propose that TEA has this effect on V438A/T449F channels because the V438A mutation produces allosteric consequences within the selectivity filter and may reorient the aromatic ring at position 449. We investigated the possibility that the blocker promotes the collapse of the outer vestibule (spring-in-the-door) in single mutant T449F channels by an electrostatic attraction between a cationic TEA and the quadrupole moments of the four aromatic rings. To test this idea, we used in vivo nonsense suppression to serially fluorinate the introduced aromatic ring at the 449 position, a manipulation that withdraws electrons from the aromatic face with little effect on the shape, net charge, or hydrophobicity of the aromatic ring. Progressive fluorination causes monotonically enhanced rates of inactivation. In further agreement with our working hypothesis, increasing fluorination of the aromatic gradually transforms the TEA effect from spring-in-the-door to foot-in-the-door. We further substantiate our electrostatic hypothesis by quantum mechanical calculations

Intent to migrate among nursing students in Uganda: Measures of the brain drain in the next generation of health professionals

Background: There is significant concern about the worldwide migration of nursing professionals from low-income countries to rich ones, as nurses are lured to fill the large number of vacancies in upper-income countries. This study explores the views of nursing students in Uganda to assess their views on practice options and their intentions to migrate. Methods: Anonymous questionnaires were distributed to nursing students at the Makerere Nursing School and Aga Khan University Nursing School in Kampala, Uganda, during July 2006, using convenience sampling methods, with 139 participants. Two focus groups were also conducted at one university. Results: Most (70%) of the participants would like to work outside Uganda, and said it was likely that within five years they would be working in the U.S. (59%) or the U.K. (49%). About a fourth (27%) said they could be working in another African country. Only eight percent of all students reported an unlikelihood to migrate within five years of training completion. Survey respondents were more dissatisfied with financial remuneration than with any other factor pushing them towards emigration. Those wanting to work in the settings of urban, private, or U.K./U.S. practices were less likely to express a sense of professional obligation and/or loyalty to country. Those who have lived in rural areas were less likely to report wanting to emigrate. Students with a desire to work in urban areas or private practice were more likely to report an intent to emigrate for financial reasons or in pursuit of country stability, while students wanting to work in rural areas or public practice were less likely to want to emigrate overall. Conclusion: Improving remuneration for nurses is the top priority policy change sought by nursing students in our study. Nursing schools may want to recruit students desiring work in rural areas or public practice to lead to a more stable workforce in Uganda.University of Washington Department of Global Healt

Clostridium difficile infection.

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota

[Aerial photography from NTS Map Sheet 072E04 - Coutts, Alberta, 06-06-1962] : [Flightline YC511-Photo 122]

072E04 - Coutts, AlbertaThis content is in the public domain in Canada and can be used without permission

Calcium Block of Single Sodium Channels: Role of a Pore-Lining Aromatic Residue

Extracellular Ca2+ ions cause a rapid block of voltage-gated sodium channels, manifest as an apparent reduction of the amplitude of single-channel currents. We examined the influence of residue Tyr-401 in the isoform rNaV1.4 on both single-channel conductance and Ca2+ block. An aromatic residue at this position in the outer mouth of the pore plays a critical role in high-affinity block by the guanidinium toxin tetrodotoxin, primarily due to an electrostatic attraction between the cationic blocker and the system of π electrons on the aromatic face. We tested whether a similar attraction between small metal cations (Na+ and Ca2+) and this residue would enhance single-channel conductance or pore block, using a series of fluorinated derivatives of phenylalanine at this position. Our results show a monotonic decrease in Ca2+ block as the aromatic ring is increasingly fluorinated, a result in accord with a cation-π interaction between Ca2+ and the aromatic ring. This occurred without a change of single-channel conductance, consistent with a greater electrostatic effect of the π system on divalent than on monovalent cations. High-level quantum mechanical calculations show that Ca2+ ions likely do not bind directly to the aromatic ring because of the substantial energetic penalty of dehydrating a Ca2+ ion. However, the complex of a Ca2+ ion with its inner hydration shell, Ca2+(H2O)6, interacts electrostatically with the aromatic ring in a way that affects the local concentration of Ca2+ ions in the extracellular vestibule