Orientation of Arabidopsis thaliana KAT1 channel in the plasma membrane.

The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K(+) channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K(+) channel, we have inserted a flag epitope in the NH(2) terminus or the S3-S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K(+) channel.

From autopoiesis to neurophenomenology: Francisco Varela's exploration of the biophysics of being

This paper reviews in detail Francisco Varela's work on subjectivity and consciousness in the biological sciences. His original approach to this [quot ]hard problem[quot ] presents a subjectivity that is radically intertwined with its biological and physical roots. It must be understood within the framework of his theory of a concrete, embodied dynamics, grounded in his general theory of autonomous systems. Through concepts and paradigms such as biological autonomy, embodiment and neurophenomenology, the article explores the multiple levels of circular causality assumed by Varela to play a fundamental role in the emergence of human experience. The concept of biological autonomy provides the necessary and sufficient conditions for characterizing biological life and identity as an emergent and circular self-producing process. Embodiment provides a systemic and dynamical framework for understanding how a cognitive self--a mind--can arise in an organism in the midst of its operational cycles of internal regulation and ongoing sensorimotor coupling. Global subjective properties can emerge at different levels from the interactions of components and can reciprocally constrain local processes through an ongoing, recursive morphodynamics. Neurophenomenology is a supplementary step in the study of consciousness. Through a rigorous method, it advocates the careful examination of experience with first-person methodologies. It attempts to create heuristic mutual constraints between biophysical data and data produced by accounts of subjective experience. The aim is to explicitly ground the active and disciplined insight the subject has about his/her experience in a biophysical emergent process. Finally, we discuss Varela's essential contribution to our understanding of the generation of consciousness in the framework of what we call his "biophysics of being".

Acute activation of Maxi-K channels (hSlo) by estradiol binding to the beta subunit.

Maxi-K channels consist of a pore-forming alpha subunit and a regulatory beta subunit, which confers the channel with a higher Ca(2+) sensitivity. Estradiol bound to the beta subunit and activated the Maxi-K channel (hSlo) only when both alpha and beta subunits were present. This activation was independent of the generation of intracellular signals and could be triggered by estradiol conjugated to a membrane-impenetrable carrier protein. This study documents the direct interaction of a hormone with a voltage-gated channel subunit and provides the molecular mechanism for the modulation of vascular smooth muscle Maxi-K channels by estrogens.

An inactive mutant of the alpha subunit of protein kinase CK2 that traps the regulatory CK2beta subunit.

Protein kinase CK2 (casein kinase 2) is a ubiquitous Ser/Thr protein kinase involved in cell proliferation. Mutation of the alpha subunit of the Xenopus laevis CK2 to change aspartic acid 156 to alanine (CK2alphaA156) resulted in an inactive enzyme. The CK2alphaA156 mutant, however, binds the regulatory subunit as measured by retention of beta on a nickel chelating column mediated by (His)6-tagged CK2alphaA156. Addition of CK2alphaA156 also caused beta to shift sedimentation in a sucrose gradient from a beta2 dimer (52 kDa) to an alpha2beta2 tetramer (130,000 kDa). CK2alphaA156 can trap the beta subunit in an inactive complex reducing the stimulation of casein phosphorylation caused by addition of beta to wild-type alpha. This competitive effect depends on the ratio of alpha/alphaA156 and on the amount of beta available. Since beta inhibits the phosphorylation of calmodulin by CK2alpha, the addition of CK2alphaA156, in this case, increases calmodulin phosphorylation by the alpha and beta combination. These results suggest that CK2alphaA156 may be a useful dominant-negative mutant that can serve to explore the multiple functions of CK2beta.