Connexin Channel Structure and Function



Connexin Channel Structure and Function

     This laboratory studies the functional properties of proteins that form ion channels through cell membranes. We study gap junction channels, which are intercellular pores through which ions and signaling molecules pass directly from cell to cell. These channels are pathways for direct electrical and molecular intercellular signaling. They are important in signal transduction, physiology, development and disease (in the nervous system they are called 'electrical synapses'). We explore, at the molecular level, mechanisms of selective molecular permeation through the pores, and mechanisms that regulate channel gating.

     Gap junction channels are formed by connexin protein. There are ~20 isoforms of connexin. Channels formed by each isoform have distinct molecular and ionic selectivities, and distinct intercellular signaling functions. Defects in each isoform cause a distinct phenotype, such as demyelination of neurons (connexin32), deafness (connexin26), cardiac malformations (connexin43), cataracts (connexin50), or increased susceptibility to cancer (connexin32). These phenotypes arise due to abnormal molecular movement through connexin pores. This molecular movement, and its control, is our focus.

    We use biophysical, biochemical and genetic approaches to explore channel structure/function. Connexin channels are studied at macroscopic and single-channel levels in reconstituted and expression systems. Genetically altered connexins are used to define permeation and modulation mechanisms. Issues we address include:

    Selective Permeation: Which molecular signals (e.g., cyclic nucleotides) pass from cell to cell are determined by the selectivity of connexin channels. We have shown that the isoform composition of connexin channels determines the size-cutoff of molecules that can permeate, and more importantly, which cyclic nucleotides and inositol phosphates can permeate. Our goal is to determine the molecular basis of the ability to distinguish among closely related second messengers; we wish to determine how the molecular selectivity is achieved and modulated.

     Open Pore Block: Molecules that enter pores and block permeation have been very useful in exploration of the structure-function of many ion channels. The absence of such reagents for connexin channels has been a major impediment for their study. We have identified and developed several classes of carbohydrate-based compounds that seem to act as specific, high-affinity open pore blockers of connexin channels. We are using these compounds as tools to explore the pore structure and function.

    Modulation: To understand the biophysics and biology of intercellular signaling, regulatory ligands for connexin channels must be found. We have identified that aminosulfonates (including taurine) and other compounds interact directly with connexin channels to modulate their activity. We are characterizing the binding sites by pharmacological, photochemical and genetic means. Derivatives of the ligands will be used to probe connexin channel structure-function.

    Ongoing projects are designed to obtain fundamental information about molecular permeation, and about the mechanisms of the modulatory sensitivities we have discovered. Photoaffinity experiments, in concert with mutagenesis, will identify the structural elements involved. The compounds we have shown to bind to connexin will be lead compounds for development of pharmacological reagents for use in structure-function studies. Through these biophysical and biochemical studies we hope to elucidate operation of connexin channels and gain insight into cellular and molecular processes that underlie intercellular signaling.

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• Bevans, C., Kordel, M., Rhee, S.K. & Harris, A.L. Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules. J. Biol. Chem. 273:2808-2816, 1998.

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• Bevans, C.G. & Harris, A.L. Regulation of connexin channels by pH: Direct action of the protonated form of taurine & other aminosulfonates. J. Biol. Chem. 274:3711-3719, 1999.

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• Harris, A.L. (2001) Emerging issues of connexin channels: Biophysics fills the gap. Quart. Rev. Biophys. 34:325-472.

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• Harris, A.L. (2002) Voltage-sensing and substate rectification: Moving parts of connexin channels. J. Gen. Physiol. 119:165-169.

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• Locke,D., Wang, L.-X., Bevans, C., Lee, Y.C. and Harris, A.L. (2003) Open pore block of connexin26 and connexin32 hemichannels by neutral, acidic and basic glycoconjugates. Cell. Commun. Adhes. 10:1-6

• Locke, D., Koreen, I.V., Liu, J.Y. and Harris, A.L. (2004) Reversible pore block of connexin channels by cyclodextrins. J. Biol. Chem. 279:22883-22892. .

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• Tao, L. and Harris, A.L. (2004) Biochemical requirements for inhibition of connexin26-containing channels by natural and synthetic taurine analogs. J. Biol. Chem. 279:38544-38554.

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• Koreen, I.V., Elsayed, W., Liu, Y.J. and Harris, A.L. (2004) Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells. Biochem. J. 383:111-119.

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