Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A – F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A – actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A – actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin – actin binding occurs in skeletal muscle sarcomeres in vivo.

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Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

SummaryKP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. From our previous structural studies, it seemed unlikely that KP4 acts by forming channels in the target cell membrane. Instead, KP4 was proposed to act by blocking fungal calcium channels, as KP4 was shown to inhibit voltage-gated calcium channels in rat neuronal cells, and its effects on fungal cells were abrogated by exogenously added calcium. Here, we extend these studies and demonstrate that KP4 acts in a reversible manner on the cell membrane and does not kill the cells, but rather inhibits cell division. This action is mimicked by EGTA and is abrogated specifically by low concentrations of calcium or non-specifically by high ionic strength buffers. We also demonstrate that KP4 affects 45 Ca uptake in U. maydis. Finally, we show that cAMP and a cAMP analogue, N 6,2 0 -O-dibutyryladenosine 3 0 :5 0 -cyclic monophosphate, both abrogate KP4 effects. These results suggest that KP4 may inhibit cell growth and division by blocking calciumregulated signal transduction pathways.

Whole body vibration (WBV) is theorized to enhance neural potentiation of the stretch reflex. The purpose of this study was to determine if WBV affects the quadriceps reflex from a patellar tendon tap. Subjects were 22 volunteers (age 23 +/- 2 yrs, ht 172.8 +/- 10.8 cm, body mass 68.6 +/- 12.3 kg). The stretch reflex was elicited from the dominant leg pre, post, and 30-min post WBV treatment. A matched control group repeated the procedure without WBV. WBV treatment consisted of 5, 1-min bouts at 26 Hz with a 1-min rest period between bouts while maintaining a standardized squatting position. Two-way ANOVAs were used to detect differences between groups over time for vastus medialis (VM) and vastus lateralis (VL) latency, EMG amplitude, electromechanical delay (EMD), and force output. No group x time interactions were detected for latency (VM; F ((2,40)) = 1.20, p = .313: VL; F ((2,40)) = 0.617, p = .544), EMG mean amplitude (VM; F ((2,40)) = 0.169, p = .845: VL; F ((2,40)) = 0.944, p = .398), EMD (VM; F ((2,40)) = 0.715, p = .495: VL; F ((2,40)) = 1.24, p = .301), or quadriceps force (F ((2,40)) = 1.11, p = .341) A single session WBV treatment does not affect the quadriceps stretch reflex in terms of timing or amplitude.