Cycling habits and other psychological variables affecting commuting by bicycle in city of Madrid

To develop effective cycling policies, decision makers and administrators should know the factors influencing the use of the bicycle for daily mobility. Traditional discrete choice models tend to be based on variables such as time and cost, which do not sufficiently explain the choice of the bicycle as a mode of transportation. Because psychological factors have been identified as particularly influential in the decision to commute by bicycle, this paper examines the perceptions of cycling factors and their influence on commuting by bicycle. Perceptions are measured by attitudes, other psychological variables, and habits. Statistical differences in the variables are established in relation to the choice of commuting mode and bicycle experience (commuter, sport���leisure, no use). Doing so enables the authors to identify the main barriers to commuting by bicycle and to make recommendations for cycling policies. Two underlying structures (factors) of the attitudinal variables are identified: direct benefits and long-term benefits. Three other factors are related to variables of difficulty: physical conditions, external facilities, and individual capacities. The effect of attitudes and other psychological variables on people���s decision to cycle to work���place of study is tested by using a logit model. In the case study of Madrid, Spain, the decision to cycle to work��� place of study is heavily influenced by cycling habits (for noncommuting trips). Because bicycle commuting is not common, attitudes and other psychological variables play a less important role in the use of bikes.

Transition to a cyclable city: policies and variables affecting cycling commuting

The growing interest in achieving the objectives of cycling policies has increased the need to know the key variables that influence the use of the bicycle for daily mobility. This paper makes a contribution in this research line by examining a varying nature of variables ��� objective and psychological - and their influence on cycling commuting in the context of a ���climber cycling city���: Vitoria-Gasteiz (Spain). Statistical differences of the variables were determined between cycling commuters and commuters by other modes. The objective variables analyzed allowed us to identify the cycling commuting profile in Vitoria-Gasteiz, but showed a small effect on cycling commuting. However, analyses on seven cycling psychological variables identified and defined, showed a higher influence, especially ���Individual capacities��� and ���Non-commuting cycling habit���. Their results allowed recommending a wide et of policy initiatives. These policy recommendations were made considering that Vitoria-Gasteiz is a ���city in transition��� towards cycling: a high level of cycling share for the Spanish contex t and the safety issue not being the main barrier for cycling. However the psychological latent variable ���Non-commuting cycling habit��� indicates that normalization of the bicycle as a mode of transport needs more progress.

Cycling Dof Factors: Molecular and functional characterization of Arabidopsis thaliana AtCDF3 and tomato (Solanum lycopersicum L.) SlCDF3 in response to abiotic stress

El tomate (Solanum lycopersicum L.) es considerado uno de los cultivos hort��colas de mayor importancia econ��mica en el territorio Espa��ol. Sin embargo, su producci��n est�� seriamente afectada por condiciones ambientales adversas como, salinidad, sequ��a y temperaturas extremas. Para resolver los problemas que se presentan en condiciones de estr��s, se han empleado una serie de t��cnicas culturales que disminuyen sus efectos negativos, siendo de gran inter��s el desarrollo de variedades tolerantes. En este sentido la obtenci��n y an��lisis de plantas transg��nicas, ha supuesto un avance tecnol��gico, que ha facilitado el estudio y la evaluaci��n de genes seleccionados en relaci��n con la tolerancia al estr��s. Estudios recientes han mostrado que el uso de genes reguladores como factores de transcripci��n (FTs) es una gran herramienta para obtener nuevas variedades de tomate con mayor tolerancia a estreses abi��ticos. Las prote��nas DOF (DNA binding with One Finger) son una familia de FTs espec��fica de plantas (Yangisawa, 2002), que est��n involucrados en procesos fisiol��gicos exclusivos de plantas como: asimilaci��n del nitr��geno y fijaci��n del carbono fotosint��tico, germinaci��n de semilla, metabolismo secundario y respuesta al fotoperiodo pero su preciso rol en la tolerancia a estr��s abi��tico se desconoce en gran parte. El trabajo descrito en esta tesis tiene como objetivo estudiar genes reguladores tipo DOF para incrementar la tolerancia a estr��s abiotico tanto en especies modelo como en tomate. En el primer cap��tulo de esta tesis se muestra la caracterizaci��n funcional del gen CDF3 de Arabidopsis, as�� como su papel en la respuesta a estr��s abi��tico y otros procesos del desarrollo. La expresi��n del gen AtCDF3 es altamente inducido por sequ��a, temperaturas extremas, salinidad y tratamientos con ��cido absc��sico (ABA). La l��nea de inserci��n T-DNA cdf3-1 es m��s sensible al estr��s por sequ��a y bajas temperaturas, mientras que l��neas transg��nicas de Arabidopsis 35S::AtCDF3 aumentan la tolerancia al estr��s por sequ��a, osm��tico y bajas temperaturas en comparaci��n con plantas wild-type (WT). Adem��s, estas plantas presentan un incremento en la tasa fotosint��tica y apertura estom��tica. El gen AtCDF3 se localiza en el n��cleo y que muestran una uni��n espec��fica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activaci��n transcripcional en ensayos de protoplastos de Arabidopsis. El dominio C-terminal de AtCDF3 es esencial para esta localizaci��n y su capacidad activaci��n, la delecci��n de este dominio reduce la tolerancia a sequ��a en plantas transg��nicas 35S::AtCDF3. An��lisis por microarray revelan que el AtCDF3 regula un set de genes involucrados en el metabolismo del carbono y nitr��geno. Nuestros resultados demuestran que el gen AtCDF3 juega un doble papel en la regulaci��n de la respuesta a estr��s por sequ��a y bajas temperaturas y en el control del tiempo de floraci��n. En el segundo cap��tulo de este trabajo se lleva a cabo la identificaci��n de 34 genes Dof en tomate que se pueden clasificar en base a homolog��a de secuencia en cuatro grupos A-D, similares a los descritos en Arabidopsis. Dentro del grupo D se han identificado cinco genes DOF que presentan caracter��sticas similares a los Cycling Dof Factors (CDFs) de Arabidopsis. Estos genes son considerados ort��logos de Arabidopsis CDF1-5, y han sido nombrados como Solanum lycopersicum CDFs o SlCDFs. Los SlCDF1-5 son prote��nas nucleares que muestran una uni��n espec��fica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activaci��n transcripcional in vivo. An��lisis de expresi��n de los genes SlCDF1-5 muestran diferentes patrones de expresi��n durante el d��a y son inducidos de forma diferente en respuesta a estr��s osm��tico, salino, y de altas y bajas temperaturas. Plantas de Arabidopsis que sobre-expresan SlCDF1 y SlCDF3 muestran un incremento de la tolerancia a la sequ��a y salinidad. Adem��s, de la expresi��n de varios genes de respuesta estr��s como AtCOR15, AtRD29A y AtERD10, son expresados de forma diferente en estas l��neas. La sobre-expresi��n de SlCDF3 en Arabidopsis promueve un retardo en el tiempo de floraci��n a trav��s de la modulaci��n de la expresi��n de genes que controlan la floraci��n como CONSTANS (CO) y FLOWERING LOCUS T (FT). En general, nuestros datos demuestran que los SlCDFs est��n asociados a funciones aun no descritas, relacionadas con la tolerancia a estr��s abi��tico y el control del tiempo de floraci��n a trav��s de la regulaci��n de genes espec��ficos y a un aumento de metabolitos particulares. ABSTRACT Tomato (Solanum lycopersicum L.) is one of the horticultural crops of major economic importance in the Spanish territory. However, its production is being affected by adverse environmental conditions such as salinity, drought and extreme temperatures. To resolve the problems triggered by stress conditions, a number of agricultural techniques that reduce the negative effects of stress are being frequently applied. However, the development of stress tolerant varieties is of a great interest. In this direction, the technological progress in obtaining and analysis of transgenic plants facilitated the study and evaluation of selected genes in relation to stress tolerance. Recent studies have shown that a use of regulatory genes such as transcription factors (TFs) is a great tool to obtain new tomato varieties with greater tolerance to abiotic stresses. The DOF (DNA binding with One Finger) proteins form a family of plant-specific TFs (Yangisawa, 2002) that are involved in the regulation of particular plant processes such as nitrogen assimilation, photosynthetic carbon fixation, seed germination, secondary metabolism and flowering time bur their precise roles in abiotic stress tolerance are largely unknown. The work described in this thesis aims at the study of the DOF type regulatory genes to increase tolerance to abiotic stress in both model species and the tomato. In the first chapter of this thesis, we present molecular characterization of the Arabidopsis CDF3 gene as well as its role in the response to abiotic stress and in other developmental processes. AtCDF3 is highly induced by drought, extreme temperatures, salt and abscisic acid (ABA) treatments. The cdf3-1 T-DNA insertion mutant was more sensitive to drought and low temperature stresses, whereas the AtCDF3 overexpression enhanced the tolerance of transgenic plants to drought, cold and osmotic stress comparing to the wild-type (WT) plants. In addition, these plants exhibit increased photosynthesis rates and stomatal aperture. AtCDF3 is localized in the nuclear region, displays specific binding to the canonical DNA target sequences and has a transcriptional activation activity in Arabidopsis protoplast assays. In addition, the C-terminal domain of AtCDF3 is essential for its localization and activation capabilities and the deletion of this domain significantly reduces the tolerance to drought in transgenic 35S::AtCDF3 overexpressing plants. Microarray analysis revealed that AtCDF3 regulated a set of genes involved in nitrogen and carbon metabolism. Our results demonstrate that AtCDF3 plays dual roles in regulating plant responses to drought and low temperature stress and in control of flowering time in vegetative tissues. In the second chapter this work, we carried out to identification of 34 tomato DOF genes that were classified by sequence similarity into four groups A-D, similar to the situation in Arabidopsis. In the D group we have identified five DOF genes that show similar characteristics to the Cycling Dof Factors (CDFs) of Arabidopsis. These genes were considered orthologous to the Arabidopsis CDF1 - 5 and were named Solanum lycopersicum CDFs or SlCDFs. SlCDF1-5 are nuclear proteins that display specific binding to canonical DNA target sequences and have transcriptional activation capacities in vivo. Expression analysis of SlCDF1-5 genes showed distinct diurnal expression patterns and were differentially induced in response to osmotic, salt and low and high temperature stresses. Arabidopsis plants overexpressing SlCDF1 and SlCDF3 showed increased drought and salt tolerance. In addition, various stress-responsive genes, such as AtCOR15, AtRD29A and AtERD10, were expressed differently in these lines. The overexpression of SlCDF3 in Arabidopsis also results in the late flowering phenotype through the modulation of the expression of flowering control genes such CONSTANS (CO) and FLOWERING LOCUS T (FT). Overall, our data connet SlCDFs to undescribed functions related to abiotic stress tolerance and flowering time through the regulation of specific target genes and an increase in particular metabolites.

Considering cycling for commuting : the role of mode familiarity : an exploration on the (circular) relation between cycling behaviours and attitudes toward cycling in Vitoria-Gasteiz, Spain

��La gente utiliza la bicicleta porque les gusta? ��O es el propio hecho de usarla la raz��n por la que les gusta hacerlo? ��O es una combinaci��n de las dos? Este tipo de preguntas reflejan un problema que se puede llamar ���el c��rculo de la consideraci��n de la bicicleta���: para poder considerar el uso de la bicicleta en el conjunto de posibles opciones a escoger, un individuo tiene que tener creencias positivas sobre ella, sobre todo en el caso de ���contextos de bajo uso���. Pero parece poco probable que se formen creencias positivas cuando hay bajos niveles de familiaridad al modo, es decir, con un bajo conocimiento de sus caracter��sticas, su funcionamiento y del imaginario asociado; al mismo tiempo, la familiaridad ir�� alcanzando niveles m��s altos conforme aumente el tiempo y la intensidad con la que se utilice la bicicleta a lo largo de la vida de los individuos. El problema parece un circulo recursivo huevo-gallina, ya que es dif��cil que alguien considere el usar la bicicleta en lugares donde su uso es una pr��ctica poco extendida. En estos lugares, y dentro del conglomerado actual de tecnolog��as, infraestructuras, reglas, pr��cticas de los usuarios y preferencias culturales que se han desarrollado alrededor del autom��vil (el actual "sistema socio-t��cnico de la movilidad urbana", Urry 2004; Geels 2005, 2012) usar la bicicleta es considerado por la mayor��a como algo dif��cil, inseguro, y anormal. Como consecuencia, los procesos de aumento de familiaridad con la bicicleta permanecen inactivos. La tesis asume la familiaridad como una fuente de informaci��n e influencia sobre las creencias positivas sobre la bicicleta. En ���contextos de bajo uso���, sin familiaridad al uso de la bicicleta, estas creencias s��lo pueden surgir de ciertos rasgos personales (afecto, valores, identidades, voluntad, etc.). Tal como han evidenciado investigaciones recientes, en estos contextos la posibilidad de considerar el uso de la bicicleta (y su eventual adopci��n), se circunscribe principalmente a los ���entusiastas���, a los que est��n dispuestos a ���ir contra corriente��� (Horton & Parkin 2012), limitando el alcance de las pol��ticas de promoci��n. La investigaci��n llevada a cabo en esta tesis ofrece un nuevo enfoque al problema del ���c��rculo de la consideraci��n de la bicicleta���. Para ello, plantea un modelo en el que se introduce a la familiaridad como un constructo que media entre el comportamiento final ���qu�� modo de transporte elige el individuo��� y el conjunto de constructos psicosociales que preceden la elecci��n modal (creencias y actitudes). La familiaridad al uso de la bicicleta se concibe como una medida de la intensidad relativa del uso de una bicicleta, real y percibida (bas��ndose en Diana & Mokhtarian 2009) que puede formarse de manera distinta seg��n sus fines (utilitarios o no utilitarios). El constructo familiaridad con el modo bicicleta est�� relacionado con la cantidad de tiempo, la intensidad y la regularidad con la que un individuo ha hecho uso de la bicicleta a lo largo de su vida. La familiaridad se concibe as�� como una condici��n que permite definir adecuadamente el contexto en el que se toman las decisiones modales de los individuos, en l��nea con investigaciones que postulan patrones de causalidad alternativos entre los procesos cognitivos de elecci��n y los comportamientos modales (Tardif 1977; Dobson et al. 1978; Golob et al. 1979; Golob 2001; Schwanen et al. 2012; Diana et al. 2009; Vij & Walker 2014). De este modo se plantea que el esquema unidireccional actitudesconductas podr��a no ser completamente valido en el caso de la consideraci��n de la bicicleta, explorando la hip��tesis que sean las propias conductas a influenciar la formaci��n de las actitudes. En esta tesis, el constructo de familiaridad se articula te��rica y metodol��gicamente, y se emplea un instrumento de dise��o transversal para contrastarlo. Los resultados de una encuesta telef��nica a una muestra representativa de 736 personas en la ciudad espa��ola de Vitoria-Gasteiz proveen evidencias que sugieren ���aunque de forma preliminar��� que la familiaridad juega un papel de mediadora en la relaci��n entre la utilizaci��n de la bicicleta y la formaci��n de las creencias y actitudes hacia el su uso. La tesis emplea mediciones para cada individuo con respecto tanto a su consideraci��n como a su familiaridad al uso de la bicicleta. ��stas mediciones se definen haciendo uso del an��lisis factorial exploratorio (AFE). Por un lado, el AFE arroja una estructura del constructo ���consideraci��n��� formada por cuatro factores, tres de ellos asociados con elementos positivos y uno con elementos negativos: (1) de c��mo el uso de la bicicleta se considera verde e inteligente (G&S); (2) sobre su car��cter agradable y adecuado (P&S); (3) sobre su eficacia como modo de transporte para ir al trabajo (E); y (4) sobre los principales inconvenientes de su uso, es decir, las dificultades impl��citas (sudoraci��n y estar expuestos a las inclemencias del tiempo) y la sensaci��n de inseguridad que genera (sentirse en riesgo de accidentes y estresarse por el tr��fico) (D&T). Por otro lado, la familiaridad al uso de la bicicleta se mide en dos distintas variables ordinales (seg��n se base en el uso utilitario o no utilitario). Como resultado, se puede hablar de que cada individuo se encuentra en una de las siguientes cuatro etapas en orden creciente hacia una familiaridad completa al modo: no familiarizados; apenas familiarizados; moderadamente familiarizados; totalmente familiarizados. El an��lisis de los datos de los cuatro grupos de sujetos de la muestra, ���definidos de acuerdo con cada una de las cuatro etapas de familiaridad definidas��� ha evidenciado la existencia de diferencias intergrupo estad��sticamente significativas, especialmente para la medida relacionada con el uso utilitario. Asimismo, las personas en los niveles inferiores de familiaridad tienen una consideraci��n menor de los aspectos positivos de la bicicleta y por el contrario presentan preocupaciones mayores hacia las caracter��sticas negativas respecto a aquellas personas que est��n m��s familiarizados en el uso utilitario. El uso, aunque espor��dico, de una bicicleta para fines utilitarios (ir de compras, hacer recados, etc.), a diferencia de no usarla en absoluto, aparece asociado a unas puntuaciones significativamente m��s altas en los tres factores positivos (G&S, E, P&S), mientras que parece estar asociado a puntuaciones significativamente m��s bajas en el factor relacionado con las caracter��sticas negativas (D&U). Aparecen resultados similares cuando se compara un uso moderado, con uno espor��dico, sobre todo con respecto a la consideraci��n de las caracter��sticas negativas. Los resultados de esta tesis est��n en l��nea con la literatura anterior que se ha basado en variables similares (por ejemplo, de Geus et al. 2008; Stinson & Bhat 2003, 2004; Hunt & Abraham 2006; y van Bekkum et al. 2011a, entre otros), pero en este estudio las diferencias se observan en un contexto de bajo uso y se derivan de un an��lisis de toda la poblaci��n de personas que se desplazan a su lugar de trabajo o estudio, lo cual eleva la fiabilidad de los resultados. La posibilidad de que unos niveles m��s altos de uso de la bicicleta para fines utilitarios puedan llevar a niveles m��s positivos de su consideraci��n abre el camino a implicaciones te��ricas y de pol��ticas que se discuten en la tesis. Con estos resultados se argumenta que el enfoque convencional basado en el cambio de actitudes puede no ser el ��nico y prioritario para lograr cambios a la hora de fomentar el uso de la bicicleta. Los resultados apuntan al potencial de otros esquemas de causalidad, basados en patrones de influencia m��s descentrados y distribuidos, y que adopten una mirada m��s positiva hacia los h��bitos de transporte, conceptualiz��ndolos como ���inteligencia encarnada y pre-reflexiva��� (Schwanen et al. 2012). Tales esquemas conducen a un enfoque m��s pr��ctico para la promoci��n del uso de la bicicleta, con estrategias que podr��an basarse en acciones de ���degustaci��n��� de su uso o de mayor ���exposici��n��� a su uso. Is the fact that people like cycling the reason for them to cycle? Or is the fact that they do cycle the reason for them to like cycling? Or is a combination of the two? This kind of questions reflect a problem that can be called ���the cycle of cycling consideration���: in order to consider cycling in the set of possible options to be chosen, an individual needs to have positive beliefs about it, especially in the case of ���low-cycling contexts���. However, positive beliefs seem unlikely to be formed with low levels of mode familiarity, say, with a low acquaintance with mode features, functioning and images; at the same time, higher levels of familiarity are likely to be reached if cycling is practised over relative threshold levels of intensities and extensively across individual life courses. The problem looks like a chicken-egg recursive cycle, since the latter condition is hardly met in places where cycling is little practised. In fact, inside the current conglomerate of technologies, infrastructures, regulations, user practices, cultural preferences that have grown around the automobile (the current ���socio-technical system of urban mobility���, Urry 2004; Geels 2005, 2012) cycling is commonly considered as difficult, unsafe, and abnormal. Consequently, the processes of familiarity forming remain disabled, and, as a result, beliefs cannot rely on mode familiarity as a source of information and influence. Without cycling familiarity, origins of positive beliefs are supposed to rely only on personal traits (affect, values, identities, willingness, etc.), which, in low-cycling contexts, confine the possibility of cycling consideration (and eventual adoption) mainly to ���cycling enthusiasts��� who are willing to ���go against the grain��� (Horton & Parkin 2012), as it results from previous research. New research conducted by author provides theoretical insights for a different approach of the cycling consideration problem in which the presence of the new construct of cycling familiarity is hypothesised in the relationship between mode choice behaviour and the set of psychosocial constructs that are supposed to precede it (beliefs and attitudes). Cycling familiarity is conceived as a measure of the real and the perceived relative intensity of use of a bicycle (building upon Diana & Mokhtarian 2009) which may be differently formed for utilitarian or non-utilitarian purposes. The construct is assumed to be related to the amount of time, the intensity and the regularity an individual spends in using a bicycle for the two distinct categories of purposes, gaining in this way a certain level of acquaintance with the mode. Familiarity with a mode of transport is conceived as an enabling condition to properly define the decision-making context in which individual travel mode choices are taken, in line with rather disperse research efforts postulating inverse relationships between mode behaviours and mode choices (Tardiff 1977; Dobson et al. 1978; Golob et al. 1979; Golob 2001; Schwanen et al. 2012; Diana et al. 2009; Vij & Walker 2014). The new construct is built theoretically and methodologically, and a cross-sectional design instrument is employed. Results from a telephone survey in a representative sample of 736 commuters in the Spanish city of Vitoria-Gasteiz, provide suggestive ���although preliminary��� evidence on the role of mode familiarity as a mediator in the relationship between cycling use and the formation of beliefs and attitudes toward cycling. Measures of both cycling consideration and cycling familiarity are defined making use of exploratory factor analysis. On the one hand, four distinct cycling consideration measures are created, based on attitude expressions on four underlying factors relating to the cycling commuting behaviour: on how cycling commuting is considered green and smart (G&S); on its pleasant and suited character (P&S); on its efficiency as a mode of transport for commuting (E); and on the main drawbacks of its use, namely the difficulties implied (sweating and being exposed to adverse weather conditions) and the sense of unsafety it generates (feeling at risk of accidents and getting stressed by traffic) (D&U). On the other hand, dimensions of cycling familiarity are measured on two distinct ordinal variables (whether based on the utilitarian or non-utilitarian use) comprising four stages to a complete mode familiarity: not familiar; barely familiar; moderately familiar; fully familiar. For each of the four stages of cycling familiarity defined, statistical significant differences are found, especially for the measure related to the utilitarian use. Consistently, people at the lower levels of cycling familiarity have a lower consideration of the positive aspects of cycling and conversely they exhibit higher concerns towards the negative characteristics than those individuals that are more familiar in utilitarian cycling. Using a bicycle occasionally for practical purposes, as opposed to not using it at all, seems associated to significant higher scores in the three positive factors (G&S, E, P&S) while it appears to be associated to significant lower scores in the factor relating with the negative characteristics of cycling commuting (D&U). A same pattern also occurs with a moderate use, as opposed to an occasional one, especially for the consideration of the negative characteristics. The results are in line with previous literature based on similar variables (e.g. de Geus et al. 2008; Stinson & Bhat 2003, 2004; Hunt & Abraham 2006; and van Bekkum et al. 2011a, among others), but in this study the differences are observed in a low-cycling context and derive from an analysis of the entire population of commuters, which rises the reliability of results.

Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: A transmembrane ��-subunit homolog

Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane �� subunit (��2) that yields inactivating MaxiK currents on coexpression with the pore-forming �� subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the ��2 subunit abolished inactivation of the �� subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle ��1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the ��2 subunit causes inactivation of the MaxiK channel �� subunit by occluding its K+-conducting pore resembling the inactivation caused by the ���ball��� peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different �� subunits.

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Unique regulatory properties of the type 2a Ca2+ channel �� subunit caused by palmitoylation

�� subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for ��2a, which differs in that it does not support prepulse facilitation of ��1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal ��1E Ca2+ channels, and is more effective in blocking inhibition of ��1E channels by G protein-coupled receptors. We show that the distinguishing properties of ��2a, rather than interaction with a distinct site of ��1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of ��2a were lost in a mutant that could not be palmitoylated [��2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three ��2 splice variants differing only in their N-termini.

R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and ��-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near ���50 mV and ���200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 ��M, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5���10 s and relaxed over 40���60 s thereafter. The [Ca2+]i increases showed a voltage threshold near ���120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in ���conditioning��� [Ca2+]i signals that regulate ion channels for stomatal function.

Acoustic overstimulation increases outer hair cell Ca2+ concentrations and causes dynamic contractions of the hearing organ

The dynamic responses of the hearing organ to acoustic overstimulation were investigated using the guinea pig isolated temporal bone preparation. The organ was loaded with the fluorescent Ca2+ indicator Fluo-3, and the cochlear electric responses to low-level tones were recorded through a microelectrode in the scala media. After overstimulation, the amplitude of the cochlear potentials decreased significantly. In some cases, rapid recovery was seen with the potentials returning to their initial amplitude. In 12 of 14 cases in which overstimulation gave a decrease in the cochlear responses, significant elevations of the cytoplasmic [Ca2+] in the outer hair cells were seen. [Ca2+] increases appeared immediately after terminating the overstimulation, with partial recovery taking place in the ensuing 30 min in some preparations. Such [Ca2+] changes were not seen in preparations that were stimulated at levels that did not cause an amplitude change in the cochlear potentials. The overstimulation also gave rise to a contraction, evident as a decrease of the width of the organ of Corti. The average contraction in 10 preparations was 9 ��m (SE 2 ��m). Partial or complete recovery was seen within 30���45 min after the overstimulation. The [Ca2+] changes and the contraction are likely to produce major functional alterations and consequently are suggested to be a factor contributing strongly to the loss of function seen after exposure to loud sounds.

Differential roles of Ca2+/calmodulin-dependent kinases in posttetanic potentiation at input selective glutamatergic pathways

The electrosensory lateral line lobe (ELL) of the electric fish Apteronotus leptorhynchus is a layered medullary region receiving electroreceptor input that terminates on basal dendrites of interneurons and projection (pyramidal) cells. The molecular layer of the ELL contains two distinct glutamatergic feedback pathways that terminate on the proximal (ventral molecular layer, VML) and distal (dorsal molecular layer) apical dendrites of pyramidal cells. Western blot analysis with an antibody directed against mammalian Ca2+/calmodulin-dependent kinase 2, �� subunit (CaMK2��) recognized a protein of identical size in the brain of A. leptorhynchus. Immunohistochemistry demonstrated that CaMK2 �� expression in the ELL was restricted to fibers and terminals in the VML. Posttetanic potentiation (PTP) could be readily elicited in pyramidal cells by stimulation of either VML or DML in brain slices of the ELL. PTP in the VML was blocked by extracellular application of a CaMK2 antagonist (KN62) while intracellular application of KN62 or a CaMK2 inhibitory peptide had no effect, consistent with the presynaptic localization of CaMK2 �� in VML. PTP in the dorsal molecular layer was not affected by extracellular application of KN62. Anti-Hebbian plasticity has also been demonstrated in the VML, but was not affected by KN62. These results demonstrate that, while PTP can occur independent of CaMK2, it is, in some synapses, dependent on this kinase.

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier���superfamily

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd ��� 0.37 ��M), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arabidopsis���thaliana

To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [��-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.

Direct interaction of G���� with a C-terminal G����-binding domain of the Ca2+ channel ��1 subunit is responsible for channel inhibition by G protein-coupled���receptors

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its ���� dimer (G����). We report below the existence of two G����-binding sites on the A-, B-, and E-type ��1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both G����-binding regions also bind the Ca2+ channel �� subunit (CC��), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in ��1E of loop 1 with that of the G protein-insensitive and G����-binding-negative loop 1 of ��1C did not abolish inhibition by G proteins, but the exchange of the ��1E C terminus with that of ��1C did. This and properties of ��1E C-terminal truncations indicated that the G����-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of G���� to this site was inhibited by an ��1-binding domain of CC��, thus providing an explanation for the functional antagonism existing between CC�� and G protein inhibition. The data do not support proposals that G���� inhibits ��1 function by interacting with the site located in the loop I���II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.