Title

Identification of pore lining amino acids and localization of gates in gap junction hemichannels

Date of Award

1998

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Physiology and Biophysics

First Committee Member

Gerhard Dahl, Committee Chair

Abstract

Gap junction channels directly connect the cytoplasm of adjacent cells and are found in almost every vertebrate and many invertebrate tissues. The essential role of gap junctions has been demonstrated in connexin knock-out experiments in mice, and the identification of human diseases associated with mutations in connexin genes. The role of gap junctions include signal conduction as well as the movement of metabolites between cells. Dye passage studies have shown that gap junction channels are able to pass molecules up to 900 Daltons in size. Gap junction channels consist of two gap junction hemichannels, one located in each membrane of adjacent cells with each hemichannel composed of six protein subunits known as connexins. The membrane topography of connexins has been determined by extensive studies using site-specific antibodies and limited proteolysis. In contrast, little is known about the correlation of structure with function e.g. the part of the protein which lines the channel and the location of the various gates. Gap junction hemichannels, the precursors of gap junctions, are known to be present in the membrane of cells and for most connexins exist only in the closed state. Recently, though, connexins have been discovered which form open hemichannels.The two connexins studied in the present thesis include a wild-type connexin (rat cx46) found in the lens of the eye and a chimeric connexin (cx32E 143) both of which form open gap junction hemichannels when expressed in Xenopus oocytes. The chimeric connexin has the backbone of cx32 with the E1 domain substituted with the E1 domain of cx43. The properties of both these connexins are similar in terms of gating. Both are closed at negative holding potentials and both are similarly affected by pH and divalent cations such as calcium and cobalt.The existence of open gap junction hemichannels has provided the opportunity to directly study the pore. We studied pore forming domains using cysteine scanning mutagenesis (CSM), a technique which has been successfully used to study the pore of other ion channels. CSM identified two reactive amino acids in the M1 domain of cx46 and cx32E143 and these two amino acids are in identical positions. The results indicate that the M1 domain in part lines the pore. One of the reactive cysteine mutants of cx46 (cx46L35C) was further studied for the mechanism of maleimidobutyryl-biocytin (MBB) inhibition. The results show that the block is steric with the possibility of an additional gating effect. Furthermore, the cysteine was accessible to thiol reagent from both sides of the membrane.By having a cysteine in the pore accessible from both sides of the membrane, it was possible to study the localization of a voltage gate and a gate closed by calcium. When the channel was closed either with voltage or with calcium, the cysteine in the pore was accessible only from the intracellular side and not the extracellular side. The results indicate that the gate or gates affected by voltage and calcium are located more extracellular to the 35 position. Voltage and calcium may be affecting the same gate or different gates.

Keywords

Biology, Cell; Biology, Animal Physiology; Biophysics, General

Link to Full Text

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