Calmodulin Mediates Ca2+-dependent Modulation of M-type K+ Channels

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 μM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 ± 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 ± 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2–5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an “IQ-like” motif present in the carboxy terminus of KCNQ2–5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic “gene gun.” Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor

Pore Helix-S6 Interactions are Critical in Governing KCNQ3 Amplitudes

Idag så anser många att vi har ett samhälle där mannen har vissa privilegier kvinnan inte har och att detta är orättvist, andra menar att det är ganska jämställt och att skillnaderna mellan könen inte är så stora. Jämställdhet är ett begrepp som uppkommer i många diskussioner och används flitigt i olika styrdokument för att skapa en bra miljö för alla människor. Intresset för debatter gällande kvinnors rättigheter och villkor har ökat de senaste åren. I bland annat politiska debatter kan vi höra om exempel som att kvinnor är underbetalda inom sina yrken. Därför har det väckt vårt intresse till att undersöka hur killar som går i gymnasiet talar om jämställdhet, feminism och normer. Vi vill ta reda på hur dagens gymnasiekillar resonerar angående dessa ämnen eftersom dessa killar är och kommer att vara en del av vårt samhälle i framtiden och deras syn på jämställdhet, feminism och normer kommer att påverka utvecklingen till ett mer jämställt samhälle. Vi använder oss av gruppintervjuer där vi intervjuar fyra olika grupper med fyra personer i varje grupp. Under studiens gång så identifierar vi återkommande mönster i materialet. Dessa teman analyserar och diskuterar vi för att få en djupare och bredare förståelse av hur killar i gymnasieåldern diskuterar kring jämställdhet samt kopplingen mellan jämställdhet och feminism. Förutom feminism kopplat till jämställdhet går vi även in på vad för sorts påverkan normer i samhället har samt vilka roll(er) könen förväntas inta, enligt informanterna. De teoretiska begrepp vi använder oss av är norm, jämställdhet, och feminism

Pore Helix-S6 Interactions Are Critical in Governing Current Amplitudes of KCNQ3 K+ Channels

AbstractTwo mechanisms have been postulated to underlie KCNQ3 homomeric current amplitudes, which are small compared with those of KCNQ4 homomers and KCNQ2/Q3 heteromers. The first involves differential channel expression governed by the D-helix within the C-terminus. The second suggests similar channel surface expression but an intrinsically unstable KCNQ3 pore. Here, we find H2O2-enhanced oligomerization of KCNQ4 subunits, as reported by nondenaturing polyacrylamide gel electrophoresis, at C643 at the end of the D-helix, where KCNQ3 possesses a histidine. However, H2O2-mediated enhancement of KCNQ4 currents was identical in the C643A mutant, and KCNQ3 H646C produced homomeric or heteromeric (with KCNQ2) currents similar to those of wild-type KCNQ3, ruling out this divergent residue as underlying the small KCNQ3 amplitudes. In KcsA, F103 in S6 is critical for pore-mediated destabilization of the conductive pathway. We found that mutations at the analogous F344 in KCNQ3 dramatically decreased the KCNQ3 currents. Total internal reflection fluorescence imaging revealed only minor differential surface expression among the wild-type and mutant channels. Homology modeling suggests that the effects of the F344 mutants arise from the disruption of the interaction between F344 and A315 in the pore helix. These data support a secondary role of the C-terminus, compared with pore helix-S6 interactions, in governing KCNQ3 current amplitudes

The Shapiro Conjecture: Prompt or Delayed Collapse in the head-on collision of neutron stars?

We study the question of prompt vs. delayed collapse in the head-on collision of two neutron stars. We show that the prompt formation of a black hole is possible, contrary to a conjecture of Shapiro which claims that collapse is delayed until after neutrino cooling. We discuss the insight provided by Shapiro's conjecture and its limitation. An understanding of the limitation of the conjecture is provided in terms of the many time scales involved in the problem. General relativistic simulations in the Einstein theory with the full set of Einstein equations coupled to the general relativistic hydrodynamic equations are carried out in our study.Comment: 4 pages, 7 figure

A Carboxy-terminal Inter-Helix Linker As the Site of Phosphatidylinositol 4,5-Bisphosphate Action on Kv7 (M-type) K+ Channels

The regulation of M-type (KCNQ [Kv7]) K+ channels by phosphatidylinositol 4,5-bisphosphate (PIP2) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of PIP2 on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential PIP2 sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of PIP2 action. Point mutants within this linker in Kv7.2 and Kv7.3 identified a conserved cluster of basic residues that interact with the lipid using electrostatic and hydrogen bonds. Homology modeling of this putative PIP2-binding linker in Kv7.2 and Kv7.3 using the solved structure of Kir2.1 and Kir3.1 channels as templates predicts a structure of Kv7.2 and 7.3 very similar to the Kir channels, and to the seven-β-sheet barrel motif common to other PIP2-binding domains. Phosphoinositide-docking simulations predict affinities and interaction energies in accord with the experimental data, and furthermore indicate that the precise identity of residues in the interacting pocket alter channel–PIP2 interactions not only by altering electrostatic energies, but also by allosterically shifting the structure of the lipid-binding surface. The results are likely to shed light on the general structural mechanisms of phosphoinositide regulation of ion channels

Collapse to Black Holes in Brans-Dicke Theory: I. Horizon Boundary Conditions for Dynamical Spacetimes

We present a new numerical code that evolves a spherically symmetric configuration of collisionless matter in the Brans-Dicke theory of gravitation. In this theory the spacetime is dynamical even in spherical symmetry, where it can contain gravitational radiation. Our code is capable of accurately tracking collapse to a black hole in a dynamical spacetime arbitrarily far into the future, without encountering either coordinate pathologies or spacetime singularities. This is accomplished by truncating the spacetime at a spherical surface inside the apparent horizon, and subsequently solving the evolution and constraint equations only in the exterior region. We use our code to address a number of long-standing theoretical questions about collapse to black holes in Brans-Dicke theory.Comment: 46 pages including figures, uuencoded gz-compressed postscript, Submitted to Phys Rev

Stimulation of κ Light-Chain Gene Rearrangement by the Immunoglobulin, µ Heavy Chain in a Pre-B-Cell Line

B-lymphocyte development exhibits a characteristic order of immunoglobulin gene rearrangements. Previous work has led to the hypothesis that expression of the immunoglobulin µ heavy chain induces rearrangement activity at the K light-chain locus. To examine this issue in more detail, we isolated five matched pairs of µ^- and endogenously rearranged µ^+ cell lines from the Abelson murine leukemia virus-transformed pro-B-cell line K.40. In four of the five µ^+ cell lines, substantial expression of µ protein on the cell surface was observed, and this correlated with an enhanced frequency of K immunoglobulin gene rearrangement compared with that in the matched µ^- cell lines. This increased K gene rearrangement frequency was not due to a general increase in the amount of V(D)J recombinase activity in the µ^+ cells. Consistently, introduction of a functionally rearranged µ gene into one of the µ^- pre-B-cell lines resulted in a fivefold increase in K gene rearrangements. In three of the four clonally matched pairs with increased K gene rearrangements, the increase in rearrangement frequency was not accompanied by a significant increase in germ line transcripts from the C_K locus. However, in the fourth pair, K.40D, we observed an increase in germ line transcription of the kappa locus after expression of µ protein encoded by either an endogenously rearranged or a transfected functional heavy-chain allele. In these cells, the amount of the germ line C_K transcript correlated with the measured frequency of rearranged K genes. These results support a regulated model of B-cell development in which µ protein expression in some way targets the V(D)J recombinase to the K gene locus