Andrew L. Harris - US grants

The gap junction channel forms a dynamically regulated aqueous pathway that mediates direct transfer of ions and small molecules between cells. The channel is composed of two integral membrane structures (each in one plasma membrane) formed of connexin protein. The intercellular communication mediated by junctional channels is considered crucial for normal development and mature function of many tissues. The channels are permeable to all known cytoplasmic second messengers. Therefore, information about how the channel functions, how it is gated, what goes through it, and how these properties can be modulated is of medical, biological and biophysical importance. The gap junction channel is difficult to study in situ because it is inaccessible for the experimental techniques and manipulations commonly applied to other channels - both ends of the channel are inside of cells. Access to the pore is via cytoplasm, so it is difficult to distinguish factors that act directly on the channel from those that act on it via intermediate cellular components. The long-term objective of this proposal is to understand the physiology and bio;physics of the gap junction channel. the approach is to incorporate the channel-forming structure into single phospholipid membranes where its properties can be studied. This proposal is to study the ion channels formed by connexin32, which forms gap junction channels between cells in vivo. Specifically, it is proposed to (1) describe and explore the permeation, gating and modulation of connexin32 channels in single phospholipid bilayers, (2) explore the effects of phosphorylation by protein kinases on the properties of connexin32 channels, and (3) explore the functional roles of specific domains of the connexin molecules. By studying the physiology of connexin channels in an experimentally accessible system, one hopes to understand the regulation of the protein that mediates junctional communication. Coupling by way of gap junction is so widespread that elucidation of this process will undoubtedly have profound effects in many areas of cellular and developmental biology.

DESCRIPTION (provided by applicant): The fundamental property of connexin channels is that they are the pathways for direct movement of cytoplasmic molecules between cells. In spite of considerable knowledge about connexin channels, the character and mechanism of their most salient property - selective permeability among signaling molecules - remains largely unknown. Which chemical signals pass though connexin channel, and how selectivity among them is achieved, are fundamental issues with far-reaching impact. We seek to define these processes. The channels formed by each of the approximately 20 varieties of connexin differ from one another with regard to molecular permeability. The importance of this specificity is demonstrated by the fact that every functional deletion of a connexin type produces a distinct pathology, such as neuronal demyelination, deafness, cardiac defects, cataracts or infertility. The pathologies that arise from altered connexin channel function must arise from abnormal molecular movement through connexin pores, whether in magnitude, regulation or molecular specificity. The proposed studies seek to define mechanism(s) by which connexin channels select among cytoplasmic signaling molecules. We also seek to further develop, characterize and utilize a class of open pore blockers of connexin channels that we have identified. The experiments address the following specific questions: Do connexin channels select among signaling molecules on the basis of specific molecular affinities and how is this achieved? What is the mechanism of pore block by cyclodextrins (CDs) and how can CDs be used to investigate pore structure? For both issues (selective permeability and block) we seek to identify the sites in the pore that are involved. Identification of such sites would be of key importance and will provide new information on the location of pore-lining parts of the protein. The work on the blockers will lead to their utilization as molecular tools to investigate connexin pore structure and function. The projects utilize a well-characterized reconstitution system to study native and heterologously-expressed connexin hemichannels to obtain information about connexin permeation selectivity and its structural basis that has been long desired, and unavailable by other means. The information obtained will inform cellular and physiological studies of intercellular communication in disease and development.

DESCRIPTION (from applicant's abstract): The physiology of intercellular Signaling through gap junctions is still a mystery. In spite of considerable progress in connexin biochemistry, and genetics, the ligands that directly control whether the channels are open or closed are unknown. Identification of fhe cytoplasmic factors that interact directly with connexin channels, and how they modulate channel activity, are fundamental unsolved issues with far-reaching impact. Gap junction channels (composed of connexin) are regulated pathways for intercellular movement of ionsn and small molecules. Their location constrains their study in situ; both ends of the pore are intracellular, inaccessible to most used to explore channel function. Since access to the channel is via cytoplasm, it is difficult to identify factors that act directly on the channel, rather than via cellular components. The long-term goal is to understand the molecular operation of this pathway of intercellular signaling. The approach is to study connexin channels in a reconstituted system where their modulation can be readily explored. Channels formed by connexin32 and connexin26 immunopurified from native tissues and expression vectors will be studied in a well-characterized system that yields information that cellular studies cannot. The experiments build on preliminary studies that have identified, for the first time, compounds that interact directly and noncovalently with connexin channels to modulate their activity. The proposed studies address the questions: What is the molecular basis for the action of protonmated aminosulfonates such as taurine on connexin chanel activity? What is the molecular basis of the high-affinity inhibition of connexin channels by the high-affinity inhibition of connexin channels by cAMP and cGMP? What parts of connexin molecules interact with these compounds? Why do the two connexins respond differently? What can be learned about connexin structure-function when derivatives of these compounds are' used as affinity reagents? By study of connexin channels in this experimentally accessible system, one hopes to understand the fudamental properties of intercellular signaling. Gap junctions are so widespread that elucidation of connexin channel activity modulation will have profound consequences throughout cellular and developmental biology. There are over 18 known connexins. In humans, genetic defects in connexin32 cause a peripheral neuropathy, and in connexin26 cause a large fraction of nonsyndromic deafness. No doubt many other syndromes anise in toto or in part from defects in connexin channel function. Such defects will result in abnormal (i.e., greater or lesser) intercellular signaling molecules. The proposed studies address how this may occur.

DESCRIPTION (provided by applicant): Mutations in the connexin26 (Cx26) gene (GJB2) are the predominant cause of inherited syndromic sensorineural deafness in humans. While most mutant connexins result in non-functional channels, some are able to form functional gap junction channels and to mediate electrical coupling (e.g., are permeable to K+ ions), yet they cause deafness. This suggests that the pathology is caused by defect(s) in intercellular movement of cytoplasmic substances other than potassium ions. It is now recognized that channels formed by each of the approximate 20 varieties of connexin differ from one another with regard to molecular permeability, and recent studies show that they can have dramatic and highly specific selectivity's among second messengers and other cytoplasmic molecules. Our hypothesis is that the deafness-causing yet functional mutants of Cx26 have altered permeability to cytoplasmic molecules, and that this defective permeability cannot be compensated for by the other connexins expressed in the inner ear. Our experimental plan is to determine the differences in selectivity of channels that contain WT and deafness-causing mutant Cx26 with regard to second messengers and other cytoplasmic molecules. In doing so, we hope to pinpoint, or narrow down the possibilities for, the specific defect in intercellular molecular movement that causes the deafness. In the inner ear almost all the channels that contain Cx26 also contain Cx30, so it is likely that the key difference in permeability is that between Cx26/Cx30 heteromeric channels that contain WT Cx26 or mutant Cx26. The proposed studies emphasize investigation studies of these heteromeric channels. The projects utilize a well-characterized reconstitution system to study native and heterologously-expressed connexin hemichannels to obtain information about connexin perm selectivity and its structural basis that has been long desired, and unavailable by other means. We will use the techniques that we have previously applied to study second messenger selectivity of other connexin channels. We anticipate that that our findings will elucidate the molecular mechanisms by which Cx26 mutants cause deafness, and identify modes of intercellular communication that are required for normal cochlear function. The information obtained will be informative regarding connexin channel function in other contexts, and will lead to insights about mechanisms of selectivity among cytoplasmic permeants.

[unreadable] DESCRIPTION (provided by applicant): This proposal explores the structure-function of the connexin channel by the use of pore blockers, focusing on two connexins in which defects cause neurological pathologies. Connexin protein forms gap junction channels, which are pathways for direct movement between cells of ions and cytoplasmic molecules, including most known second messengers. Serious pathologies arise from connexin defects, including demyelination, deafness, skin disorders and cataracts, depending on the connexin isoform affected. Despite acute biological and medical interest, the mechanism of the defining property of connexin channels - the ability of the pore to mediate selective molecular permeability between cells - has not been elucidated. Investigation of the structure and function of connexin pores has been hampered by the absence of molecular reagents that enter and bind in the pore. This class of reagents ("pore blockers") has been of inestimable value in elucidation of the structure-function of permeation of other channels. This project applies newly identified connexin pore blockers to investigate the connexin pore, and to thus obtain key information that has been long desired. Preliminary studies have identified two classes of carbohydrate-based connexin pore blockers, and established their feasibility as investigational tools of connexin channels. For the first class, novel glycinamide derivatives of aminobenzoic acid glycinamides (ABGs) were designed and conjugated to a size-indexed set of maltosaccharides. The resulting ABG-glycoconjugates act as reversible, high affinity blockers of molecular permeation through connexin pores in a size- and connexin-specific manner, whereas the maltosaccharides or the ABGs alone do not block. The second class of blockers are cyclodextrins (CDs), which are cyclized glucosaccharides. They also block connexin pores in a reversible, size-specific manner. For both classes of blockers, the correlation between the size of the molecule required for block and the relative width of the pore (determined using a size-indexed series of permeable sugars) indicate that their site of action is within the pore. The proposed studies build on this work. Aim 1 investigates the chemical determinants and mechanism of the intra-pore binding of the ABG- glycoconjugates. Aim 2 initiates application of the ABG-sugars to the study of connexin channels. Aim 3 utilizes naturally-occurring and modified CDs to probe the connexin pore. The projects primarily utilize a well- characterized reconstitution system to study heterologously-expressed connexin channels to obtain information unavailable by other means. It is anticipated that the development and application of these pore blockers will enable and inform the biophysical and cellular studies required to define the molecular mechanisms of intercellular communication in development and disease. In the present proposal, this analysis will be applied to Cx32 and Cx26, defects in which cause X-linked Charcot-Marie-Tooth disease neuropathy and sensorineural deafness, respectively. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Connexin proteins form the gap junction channels that mediate direct intercellular molecular communication crucial in development, physiology and response to trauma/inflammation/disease. Mutations in connexins cause human pathologies. Elucidation of the molecular mechanisms that regulate connexin channel function is essential for understanding their roles in human physiology and pathophysiology, and to identify targets for translational and basic science studies. The long-term goal of this project is to understand the cytosolic inter-domain interactions of connexin proteins that control channel gating. In connexin43 (Cx43), interactions between the cytoplasmic loop (CL) and the C-terminal domain (CT) is crucially involved physiological function of the channel and is a target for cardiovascular therapies. However, the roles and mechanisms of CL-CT interactions in modulation of other connexin channels, just as likely to be biomedically important, have not been explored. We have found that Cx26 channels are modulated by CL-CT interactions. Cx26 and Cx43 are representative members of the two largest families of connexins. Though structurally analogous, the effects of CL-CT interaction on Cx26 channel function seem to be fundamentally different from those in Cx43, suggesting that CL-CT interaction is a common modulatory mechanism in connexins, yet operates in connexin-specific ways. We propose to elucidate the molecular mechanisms of CL-CT control of channel function using Cx26 and its closely related isoform Cx32. Cx26 is the only connexin channel for which there is a high-resolution structure, making it the cornerstone for structure-based studies of all connexin channels. The proposed studies explore the basis and mechanisms of CL-CT interactions and channel properties they modulate, using strategies successfully applied to other channels, including macroscopic and single channel recordings, use of competing peptides, engineered Cys linkages and mutational analysis. The experiments utilize intact channels, complemented by peptide NMR. We propose to (a) determine the involvement of CL-CT interactions in channel gating, (b) identify the sites of CL-CT interactions, and (c) determine how CL-CT interaction and its effects are altered by mutations that cause human disease. Cx26 and Cx32 are widely distributed in the body. Cx26 mutations cause over half the inherited non-syndromic sensorineural deafness worldwide, and serious disfiguring skin disorders. Cx32 mutations cause a peripheral demyelination. Significantly, in both connexins many disease-causing mutations are in the CL and CT domains. Both connexins are involved in a wide range of pathological and physiological processes, so understanding the basis for their dysregulation has broad biomedical implications, for these connexins and others. The large number of disease causing mutations and the extensive experience of both PIs in studying gating and permeability of Cx26 and Cx32 channels provide a basis for productive, informative investigation of CL-CT interactions, how they are altered by pathological mutations and the effects on channel function.

Connexins are integral membrane proteins that oligomerize to form gap junction channels. Gap junctions composed of Cx43 mediate electrical coupling and impulse propagation in the normal working myocardium. In the failing heart, Cx43 remodeling (decreased expression, loss at intercalated discs, increased presence at lateral membranes) contributes to ventricular arrhythmias. However, the failing heart also aberrantly upregulates expression of Cx45 in ventricles, where it is normally at very low levels. This greatly enhances the propensity for arrhythmias, logically due to the low conductance and high voltage-sensitivity of Cx45 channels relative to Cx43. Crucially, the deleterious effect of Cx45 at the intercalated discs is likely amplified by the propensity of Cx45 to form heteromeric channels with Cx43, in which it has a dominant effect on the function of the resulting channels. Unfortunately, little is known about the mechanisms that drive Cx45 presence at intercalated discs or about the determinants of the functional properties of Cx45 that make its presence at ventricular intercalated discs dangerous. Studies proposed in Specific Aims 1 and 2 address novel mechanisms by which phosphorylation of the Cx45 carboxyl terminal (Cx45CT) domain modulates Cx45 protein partner interactions to increase or decrease gap junction intercellular communication in vitro and in vivo (and the differences from effects on Cx43). Specific Aim 3 focuses on determining how a recently discovered high-affinity protein-protein interaction of the Cx45CT, dimerization, affects the channel functional properties. The significance of this proposal is that discovery of how phosphorylations and interactions of the CT domain can be modulated would enable strategies to ameliorate pathological alterations of connexins the failing heart and elsewhere.