Functional modification of ion channels by reagents that interact with specific amino acids may provide clues regarding the channel structure-function relationship (Spires & Begenisich, 1992; Varnum et al. 1995). As more information about the primary structure of ion channels becomes available, chemical modification methods may help map the location of essential residues in ion channel function. Modification of channel proteins may either reduce ionic current (Drews & Rack, 1988), or cause persistent channel activation (Varnum et al. 1995).
In the present study, we have identified the amino acid residues crucial for normal Cl− channel function in bovine tracheal epithelial cells. Bovine cells have frequently been used to study transepithelial ion movement, since they express DIDS-sensitive Cl− channels and amiloride-sensitive Na+ channels (Durand et al. 1986; Valdivia et al. 1988). The predominant Cl− channel has a linear current-voltage relationship with a conductance of ∼71 pS, it is activated by cAMP-dependent phosphorylation but not by Ca2+ and accounts for more than 80 % of the total Cl− conductance (Valdivia et al. 1988). Its halide permeability sequence (Cl− > Br− I−) resembles a mid-range Eisenman sequence for anions (Wright & Diamond, 1977), indicating that permeation through the pore is a combination of dehydration and weak interaction with an internal binding site.
Here we report that the activity of the epithelial Cl− channel is significantly increased by modification of lysine residues located in the extracellular domain of the channel.
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Our results show that amino group-specific reagents increase the activity of the 71 pS Cl− channel. Since imidoesters such as MA and DMA are regarded as truly lysine-specific reagents (Makoff & Malcolm, 1981), this suggests that lysine residues are involved in control of Cl− channel activity. The possibility that the observed effects are due to modification of the amino group of membrane phospholipids is less likely as similar results were obtained in patch clamp and Cl− flux studies. Such phospholipids would have to be considered an integral part of the channel that is preserved after reconstitution into lipid membranes.
The fact that the 71 pS Cl− channel is regulated by PKA-dependent phosphorylation allowed us to demonstrate the orientation of the channels after reconstitution into liposomes. Dephosphorylation of channel proteins before reconstitution reduced Cl− flux, confirming that PKA-dependent Cl− channels contribute the main fraction of Cl− flux. However, dephosphorylation of channel proteins after reconstitution into liposomes had no significant effect on Cl− flux. This suggests that phosphorylation sites on channel proteins are not accessible to alkaline phosphatase after reconstitution, and are probably located inside the liposome. This conclusion is in agreement with other experimental observations suggesting that proteins are incorporated into lipid bilayers in a unidirectional orientation determined solely by protein features (Brunen & Engelhardt, 1993). Additionally, studies of protein orientation in liposomes indicated that between 75 and 95 % of all proteins preserved their original orientations in the cell membrane (Gennis, 1989). Therefore we assumed that channel orientations in vesicles were similar to their original orientations in the native cells.
The different effects of DMA and DTSSP on channel function in excised patches is probably related to the fact that DMA is membrane permeant (Han et al. 1984), while DTSSP is membrane impermeant (Varnum et al. 1995). This suggests that the modified lysine residues are located on the extracellular surface of the cells. It is interesting to note that while Cl− flux was significantly reduced by 50 μm H2-DIDS in liposomes containing native proteins, this blocker was ineffective after modification of Cl− channels with DMA. This suggests either that the binding site for H2-DIDS after DMA modification is occupied by DMA, or that DMA modification causes conformational changes in channel proteins that prevent the binding of H2-DIDS.
DMA and DTSSP are bifunctional reagents that introduce cross-linking of lysine residues, and are often used to identify spatial arrangements in proteins (Han et al. 1984). Both reagents increased the Cl− flux, suggesting that the Cl− channel has two lysine residues amenable to cross-linking reactions. However, the mono-functional imidoester MA exerted a similar effect, indicating that only one lysine residue has to be modified to activate the Cl− channel.
Patch clamp studies have shown that the channel open probability, but not its conductance, increased after DMA treatment. This indicates that the modification site is an important part of the channel gating mechanism and is probably distant to the channel permeation pathway. Kinetic analysis of channel activity indicated that the distributions of open and closed durations were best approximated by a sum of two components, suggesting that the channel has a minimum of two open and two closed states (Korn & Horn, 1988). After DMA modification, both distributions were also best approximated by a sum of two components but the fitting parameters were significantly changed. As a result, the channel spent significantly more time in the open state than before DMA treatment. This effect is similar to the stimulation of CFTR Cl− channels by covalent modification of the regulatory domain with NEM (Cotten & Welsh, 1997). These authors concluded that the conformational change of this domain may be a key feature that regulates channel activity.
Full understanding of ion channel function requires molecular information on channel sequence and structure and their relationship to function. Recently, Cunningham et al. (1995) cloned a putative Ca2+-dependent Cl− channel from bovine trachea. The lack of amino acid sequence information in the case of the 71 pS Cl− channel prevents us from relating observed effects of amino group modification to precise locations on the channel protein. However, several studies have shown that amino acid residues involved in channel gating may be located in the extracellular domain. For example, Lopez-Barneo et al. (1993) have shown that mutations near the outer mouth of the Shaker K+ channel pore produced drastic changes in C-type inactivation kinetics. The authors proposed a ‘foot-in-the-door’ model of channel gating, which could reflect the interaction of ions with amino acids in the channel pore.
Modification of amino groups by imidoesters and DTSSP produces large kinetic changes in channel function, suggesting that these residues may be important candidates for site-directed mutagenesis studies. The fact that these residues are accessible from the extracellular side may play an important role in manipulation of Cl− channels in intact cells. Therefore, our work may have clinical implications in diseases characterized by reduced ion channel permeability, such as cystic fibrosis. Although the lack of protein specificity of amino group-specific reagents prohibits their therapeutic use, their stimulatory effects may suggest other pharmacological approaches by which channel activity can be augmented through modification of the extracellular domain.