Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel expressed predominantly in peripheral nociceptors. By detecting and integrating diverse noxious thermal and chemical stimuli, and as a result of its sensitization by inflammatory mediators, the TRPV1 receptor plays a key role in inflammation-induced pain. Activation of TRPV1 leads to a cascade of pro-nociceptive mechanisms, many of which still remain to be identified. Here, we report a novel effect of TRPV1 on the activity of the potassium channel KCNQ2/3, a negative regulator of neuronal excitability. Using ion influx assays, we revealed that TRPV1 activation can abolish KCNQ2/3 activity, but not vice versa, in human embryonic kidney (HEK)293 cells. Electrophysiological studies showed that coexpression of TRPV1 caused a 7.5-mV depolarizing shift in the voltage dependence of KCNQ2/3 activation compared with control expressing KCNQ2/3 alone. Furthermore, activation of TRPV1 by capsaicin led to a 54% reduction of KCNQ2/3-mediated current amplitude and attenuation of KCNQ2/3 activation. The inhibitory effect of TRPV1 appears to depend on Ca2+ influx through the activated channel followed by Ca2+-sensitive depletion of phosphatidylinositol 4,5-bisphosphate and activation of protein phosphatase calcineurin. We also identified physical interactions between TRPV1 and KCNQ2/3 coexpressed in HEK293 cells and in rat dorsal root ganglia neurons. Mutation studies established that this interaction is mediated predominantly by the membrane-spanning regions of the respective proteins and correlates with the shift of KCNQ2/3 activation. Collectively, these data reveal that TRPV1 activation may deprive neurons from inhibitory control mediated by KCNQ2/3. Such neurons may thus have a lower threshold for activation, which may indirectly facilitate TRPV1 in integrating multiple noxious signals and/or in the establishment or maintenance of chronic pain.

Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.

A physical model of selective “ion binding” in the L-type calcium channel is constructed, and consequences of the model are compared with experimental data. This reduced model treats only ions and the carboxylate oxygens of the EEEE locus explicitly and restricts interactions to hard-core repulsion and ion–ion and ion–dielectric electrostatic forces. The structural atoms provide a flexible environment for passing cations, thus resulting in a self-organized induced-fit model of the selectivity filter. Experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed using equilibrium Monte Carlo simulations in the grand canonical ensemble. The model pore rejects alkali metal ions in the presence of biological concentrations of Ca2+ and predicts the blockade of alkali metal ion currents by micromolar Ca2+. Conductance patterns observed in varied mixtures containing Na+ and Li+, or Ba2+ and Ca2+, are predicted. Ca2+ is substantially more potent in blocking Na+ current than Ba2+. In apparent contrast to experiments using buffered Ca2+ solutions, the predicted potency of Ca2+ in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with Ca2+ for the pore. These experiments depend on the problematic estimation of Ca2+ activity in solutions buffered for Ca2+ and pH in a varying background of bulk salt. Simulations of Ca2+ distribution with the model pore bathed in solutions containing a varied amount of Li+ reveal a “barrier and well” pattern. The entry/exit barrier for Ca2+ is strongly modulated by the Li+ concentration of the bath, suggesting a physical explanation for observed kinetic phenomena. Our simulations show that the selectivity of L-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. The reduced system selects for the cation that delivers the largest charge in the smallest ion volume.

The experiments indicate that the protoplasm of Nitella consists of an aqueous layer W with an outer non-aqueous surface layer X and an inner non-aqueous surface layer Y. The potential at Y is measured by the magnitude of the action curve and the potential at X by the distance from the top of the action curve to the zero line. These potentials appear to be due chiefly to diffusion potentials caused by the activity gradients of KCl across the non-aqueous layers X and Y. The relative mobilities of K+ and Cl- in X and in Y can be computed and an estimate of the activity of KCl in W can be made. In the complete resting state the mobilities of K+ and Cl- in X are not very different from those in Y. The action curve is due to changes in Y which suddenly becomes very permeable, allowing potassium to move from the sap across Y into W, and thus losing its potential. A gradual loss may be due to changes in ionic mobility in Y. When recovery is incomplete and Y has not yet regained its normal potential a stimulus may cause a loss of the potential at Y giving an action curve of small magnitude. The magnitude may vary in successive action curves giving what is called a complex pattern in contrast to the simple pattern observed when recovery is complete and all the action curves are alike. Complex patterns occur chiefly in cells treated with reagents. Untreated cells usually give simple patterns. A variety of complex action patterns is discussed. It is evident that the cells of Nitella show much more variation than such highly specialized cells as muscle and nerve which give stereotyped responses. In some cases it may be doubtful whether the all-or-none law holds.

1. Crystalline hexokinase has been isolated from baker's yeast. 2. Crystalline hexokinase is a protein of albumin type of a molecular weight of 96,000. Its isoelectric point is at about pH 4.8. 3. The method of isolation consists in separating the proteins of an aqueous extract of toluene-treated yeast by means of fractional precipitation with ammonium sulfate and with alcohol. 4. The procedure involves also the separation of several crystalline proteins, including one yellow crystalline protein, which do not possess hexokinase activity. The biological and the physicochemical properties of these proteins are still under investigation. 5. The crystallization of hexokinase proceeds at about 5°C. in the presence of ammonium sulfate and dilute phosphate buffer pH 7.0. 6. Crystalline hexokinase becomes relatively pure after 2 or 3 recrystallizations as tested by solubility, sedimentation in the ultracentrifuge, and electrophoresis. The enzymatic activity remains constant on repeated crystallization. 7. The enzymatic activity is associated with the protein nature of the material. Inactivation is accompanied by denaturation of the protein. 8. Crystalline hexokinase is relatively stable when stored in the form of crystalline filter cake. Solutions of hexokinase in dilute buffers are most stable at pH 5.0. 9. Crystalline hexokinase requires the presence of magnesium ions for its catalytic activity.

Rapid local cooling of mammalian nerve sets up a discharge which is preceded by a local temperature potential, the cooled region being electronegative relative to a normal portion of the nerve. Heating the nerve locally above its normal temperature similarly makes the heated region electronegative relative to a region at normal temperature, and again a discharge is set up from the heated region. These local temperature potentials, set up by the nerve itself, are held to serve as "generator potentials" and the mechanism found is regarded as the prototype for temperature end organs.

The results of the study of the inhibiting effect of neutral salts upon the clotting tendency of fibrinogen by thrombin may be summarised as follows: Salts like NaCl and KCl inhibit only weakly. Salts of the same cation (K•) with monovalent anions of different ionic radius are the more active the larger the anion (Cl',Br',I'). Salts of the same cation with anions of different valency are the more active the higher the charge of the anion (1–1 <1–2 <1–3 <1–4). Salts with the same anion with cations of different valency show stronger inhibition in the case of cations of higher charge (K•,Na• < Mg••, Ca••, Sr••, Ba••). Salts with the same anion and cations of the same charge, but of different radius, are the more active the larger the cation (but with an inversion between Mg•• and Ca•• in the series of the alkali earths, which is not infrequent in biocolloids). These results show that the clotting of fibrinogen with thrombin is, at least partly, caused by a coacervation process, due to electrostatic attraction between positive and negative groups. Its nature and localisation will be dealt with in the next paper of this series.

The flicker contour for the house sparrow Passer domesticus is duplex, corresponding to the presence of both rods and cones in the retina. The presence of the pecten brings about changes in the "cone" part of the contour when the light-time in the flash cycle is varied. These changes are of the same sort as those we have already described for the visually simplex zebra finch, and for man provided with an artificial "pecten shadow." The changes are such as to greatly enhance flicker acuity for small dark-times (moving stripe technique). The form of the scotopic part of the duplex contour (also as in the case with man) gives no evidence that rod excitation is specifically influenced by the presence of the pecten. The changing integration of "rod" and "cone" effects as the light-time fraction is altered provides another means of testing the theory used for the analytical separation of the two components of the duplex flicker contour.

1. When there is projected on the retina (man, monocularly) the shadow of a grid which forms a visual field in several distinct pieces (not including the fovea in the present tests), the ordinary properties of the flicker recognition contour (F vs. log I) as a function of the light-time cycle fraction (t(L)) can be markedly disturbed. In the present experiments flicker was produced by the rotation of a cylinder with opaque vertical stripes. In the absence of such a grid shadow the "cone" segments of the contours form a set in which F(max.) and the abscissa of inflection are opposite but rectilinear functions of t(L), while the third parameter of the probability integral (sigma'(log I)) remains constant. This is the case also with diverse other animals tested. In the data with the grid, however, analysis shows that even for low values of t(L) (up to 0.50) there occurs an enhancement of the production of elements of neural effect, so that F(max.) rises rather than falls as ordinarily with increase of t(L), although sigma'(log I) stays constant and hence the total number of acting units is presumed not to change. This constitutes valid evidence for neural integration of effects due to the illumination of separated retinal patches. Beginning at t(L) = 0.75, and at 0.90, the slope of the "cone" curve is sharply increased, and the maximum F is far above its position in the absence of the grid. The decrease of sigma'(log I) (the slope constant) signifies, in terms of other information, an increase in the number of acting cone units. The abscissa of inflection is also much lowered, relatively, whereas without the grid it increases as t(L) is made larger. These effects correspond subjectively to the fact that at the end-point flicker is most pronounced, on the "cone" curve, along the edges of the grid shadow where contrast is particularly evident with the longer light-times. The "rod" portion of the F - log I contour is not specifically affected by the presence of the grid shadow. Its form is obtainable at t(L) = 0.90 free from the influence of summating "cone" contributions, because then almost no overlapping occurs. Analysis shows that when overlapping does occur a certain number of rod units are inhibited by concurrent cone excitation, and that the mean contribution of elements of neural action from each of the non-inhibited units is also reduced to an extent depending on the degree of overlap. The isolated "rod" curve at t(L) = 0.90 is quite accurately in the form of a probability integral. The data thus give a new experimental proof of the occurrence of two distinct but interlocking populations of visual effects, and experimentally justify the analytical procedures which have been used to separate them. 2. The changing form of the F - log I contour as a function of t(L), produced in man when the illuminated field is divided into parts by a shadow pattern, is normally found with the bird Taeniopygia castenotis (Gould), the zebra finch. The retina has elements of one general structural type (cones), and the F - log I contour is a simplex symmetrical probability integral. The eye of this bird has a large, complex, and darkly pigmented pecten, which casts a foliated shadow on the retina. The change in form of the F - log I curve occurs with t(L) above 0,50, and at t(L) = 0.90 is quite extreme. It is more pronounced than the one that is secured in the human data with the particular grid we have used, but there is no doubt that it could be mimicked completely by the use of other grids. The increase of flicker acuity due to the pecten shadow is considerable, when the dark spaces are brief relative to the light. The evidence thus confirms the suggestion (Menner) drawn from comparative natural history that the visual significance of the avian pecten might be to increase the sensory effect of small moving images. It is theoretically important that (as in the human experiment) this may be brought about by an actual decrease of effective retinal area illuminated. It is also significant theoretically that despite the presence of shadows of pecten or of grid, and of the sensory influences thus introduced, the probability integral formulation remains effective.