LACK OF CONVENTIONAL ATPASE PROPERTIES IN CFTR CHLORIDE CHANNEL GATING

CFTR shares structural homology with the ABC transporter superfamily o f proteins which hydrolyze ATP to effect the transport of compounds ac ross cell membranes. Some superfamily members are characterized as P-t ype ATPases because ATP-dependent transport is sensitive to the presen ce of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrol ytic activity in channel,eating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride chan nel activity under conditions known to inhibit the activity of ATPases ; i.e., in the absence of divalent cations and in the presence of a va riety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, r educed both the opening and closing rates of CFTR suggesting that Mg2 plays a modulatory role in channel gating. However, channels continue d to both open and close showing that Mg2+ is not an absolute requirem ent for channel activity. The nonselective P-type ATPase inhibitor, va nadate, did not alter the gating of CFTR when used at concentrations w hich completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR , but did not affect the opening of the channel. As expected, more sel ective P-type (Sch28080, ouabain), V-type (bafilomycin Al, SCN-) and F -type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhib ition profile with other ATPases and channel gating occurs in the appa rent absence of hydrolysis, although with altered kinetics. Vanadate i nhibition of channel closure might suggest that a hydrolytic step is i nvolved although the requirement for a high concentration raises the p ossibility of previously uncharacterized effects of this compound. Mos t conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for this transition state analogue is considerably different than that of other ABC transporters.