Substituted cysteine scanning in D1-S6 of the sodium channel hNav1.4 alters kinetics and structural interactions of slow inactivation.

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.

Plasma cell-free mitochondrial DNA declines in response to prolonged moderate aerobic exercise.

Increased plasma cell-free mitochondrial DNA (cf-mDNA), a damage-associated molecular pattern (DAMP) produced by cellular injury, contributes to neutrophil activation/inflammation in trauma patients and arises in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we examined cf-mDNA response to prolonged moderate aerobic exercise. Seven healthy moderately trained young men (age = 22.4 ± 1.2) completed a treadmill exercise trial for 90 min at 60% VO2 max and a resting control trial. Blood was sampled immediately prior to exercise (0 min = baseline), during (+18, +54 min), immediately after (+90 min), and after recovery (R40). Plasma was analyzed for cf-mDNA, IL-6, and lactate. A significant difference in cf-mDNA response was observed between exercise and control trials, with cf-mDNA levels reduced during exercise at +54 and +90 (with or without plasma volume shift correction). Declines in cf-mDNA were accompanied by increased lactate and followed by an increase in IL-6, suggesting a temporal association with muscle stress and inflammatory processes. Our novel finding of cf-mDNA decline with prolonged moderate treadmill exercise provides evidence for increased clearance from or reduced release of cf-mDNA into the blood with prolonged exercise. These studies contrast with previous investigations involving exhaustive short-term treadmill exercise, in which no change in cf-mDNA levels were reported, and contribute to our understanding of differences between exercise- and trauma-induced inflammation. We propose that transient declines in cf-mDNA may induce health benefits, by reducing systemic inflammation.

Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing.

Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRß) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is similar for both genes and consistent with results at the TCRß locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (Pr nucleotides) correspond mostly to single-base additions consistent with normal TdT activity.

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na(+) channels.

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na(+) channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na(+) current (I(Na)). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I(Na). Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4min), there is little effect on I(Na). However, with longer exposures (4-8min), there is a large decrease in I(Na), but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) - MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na(+) channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.

Relative resistance to slow inactivation of human cardiac Na+ channel hNav1.5 is reversed by lysine or glutamine substitution at V930 in D2-S6.

Transmembrane segment 6 is implicated in slow inactivation (SI) of voltage-gated Na(+) channels (Na(v)s). To further study its role and understand differences between SI phenotypes of different Na(v) isoforms, we analyzed several domain 2-segment 6 (D2-S6) mutants of the human cardiac hNa(v)1.5, which is relatively resistant to SI. Mutants were examined by transient HEK cell transfection and patch-clamp recording of whole cell Na(+) currents. Substitutions with lysine (K) included N927K, V930K, and L931K. We show recovery from short (100 ms) depolarization to 0 mV in N927K and L931K is comparable to wild type, whereas recovery in V930K is delayed and biexponential, suggesting rapid entry into a slow-inactivated state. SI protocols confirm enhanced SI phenotype (rapid development, hyperpolarized steady state, slowed recovery) for V930K, contrasting with the resistant phenotype of wild-type hNa(v)1.5. This enhancement, not found in N927K or L931K, suggests that the effect in V930K is site specific. Glutamine (Q) substituted at V930 also exhibits an enhanced SI phenotype similar to that of V930K. Therefore, K or Q substitution eliminates hNa(v)1.5 resistance to SI. Alanine (A) or cysteine (C) substitution at V930 shows no enhancement of SI, and in fact, V930A and V930C, as well as L931K, exhibit a resistance to SI, demonstrating that characteristics of specific amino acids (e.g., size, hydrophobicity) differentially affect SI gating. Thus V930 in D2-S6 appears to be an important structural determinant of SI gating in hNa(v)1.5. We suggest that conformational change involving D2-S6 is a critical component of SI in Na(v)s, which may be differentially regulated between isoforms by other isoform-specific determinants of SI phenotype.

Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel.

To examine conformational changes during slow inactivation involving domain 2-segment 6 (D2-S6) of human cardiac Na(+) channel (hNav1.5), we applied the substituted-cysteine accessibility method (SCAM) using methanethiosulfonate ethylammonium (MTSEA). We substituted cysteine (C) for native valine (V) at position 930 of D2-S6 in the MTSEA-resistant hNav1.5 mutant C373Y to produce the double mutant C373Y-V930C. Whole-cell Na(+) currents were recorded using patch-clamp techniques in transiently transfected HEK cells. In C373Y-V930C, we find that MTSEA (1.5 mM) applied in the closed state (-160 mV) has no significant effect on whole-cell Na(+) current, while MTSEA applied in the slow-inactivated state (prolonged depolarization at 0 mV) decreases current. We propose that D2-S6 in hNav1.5 undergoes molecular rearrangement during slow inactivation exposing the side chain of residue 930 such that it becomes accessible to modification by MTSEA.

Partial reconstitution of V(D)J rearrangement and lymphocyte development in RAG-deficient mice expressing inducible, tetracycline-regulated RAG transgenes.

Previously, we described a tetracycline-based autoregulatory system for inducible gene expression in mammalian cells and transgenic mice [Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6522]. We have tested the ability of this system to drive functional expression in vivo of the V(D)J recombination activating genes, RAG1 and RAG2. In induced transgenic mice, transgenic RAG1 and RAG2 mRNA is observed in thymus and spleen, and expression of both transgenes on the RAG1 or RAG2 knockout backgrounds allows partial, inducible, lymphocyte reconstitution. In thymus and peripheral lymphoid organs of reconstituted animals, cells expressing CD4 and/or CD8 on their surface, also express CD3 and TCR beta chain. In these animals, V(D)J rearrangements are detected in thymus, lymph nodes, and spleen at the TRB locus, and in thymus and lymph nodes at the TRD locus. At the TRA locus, broken ends at V(D)J recombination signals are detected only in thymus, as are reciprocal signal joint products derived from deletional rearrangement. T cell reconstitution occurs in these animals whether they are induced in utero during development, or shortly after birth. A low level of B cell reconstitution is also observed. B220+IgM+ cells are observed in spleen only in induced animals, and rearrangements at IGH and IGK loci are detected in bone marrow and spleen. Broken signal ends at the IGK locus, are not detected in peripheral lymphoid organs. Inducible reconstitution of normal levels of serum immunoglobulin, including heavy chain class switch isotype variants is also observed in these animals. Further, these transgenes do not appear to interfere with lymphocyte development mediated by functionally rearranged TRB chain or IGH chain transgenes in RAG-deficient animals. These mice provide a unique system for the inducible activation of V(D)J recombination and the development of primary lymphocytes.

Inducible, reversible hair loss in transgenic mice.

Telogen effluvium is a common type of hair loss. Although the morphological changes associated with telogen effluvium have been well characterized, the underlying molecular mechanisms remain unknown, and no animal models have been developed. We report here that inducible transgenic mice expressing high levels of the transcription factor, tTA (tetracycline transactivator), plus a reporter luciferase gene, show a reversible hair loss phenotype. Skin of these mice exhibits an increase in the number of hair follicles at the telogen phase, but a decreased number of follicles at the anagen phase. These changes resemble skin pathology seen in patients with telogen effluvium, which suggests that the inducible transgenic mice may be useful as a model for this disorder. Moreover, since overexpression of several other transgenes failed to cause skin pathology, the present findings also indicate types of molecular abnormalities that may cause reversible hair loss.