FUNCTIONAL CONSEQUENCES OF SULFHYDRYL MODIFICATION IN THE PORE-FORMING SUBUNITS OF CARDIOVASCULAR CA2+ AND NA+ CHANNELS

The structure and function of many cysteine-containing proteins critic ally depend on the oxidation state of the sulfhydryl groups. In such p roteins, selective modification of sulfhydryl groups can be used to pr obe the relation between structure and function. We examined the effec ts of sulfhydryl-oxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit s mooth muscle L-type Ca2+ channel and the human cardiac tetrodotoxin-in sensitive Na+ channel. The known sequences of the channels suggest the presence of three or four cysteine residues within the putative pores of Ca2+ or Na+ channels, respectively, as well as multiple other cyst eines in regions of unknown function. We determined the effects of sul fhydryl modification on Ca2+ and Na+ channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp te chnique. Within 10 minutes of exposure to 2,2'-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca2+ current wa s reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The eff ect could be readily reversed by 1,4-dithiothreitol (an agent that red uces disulfide bonds). Similar results were obtained by using the hydr ophilic sulfydryl-oxidizing agent thimerosal. The effects were Ca2+-ch annel specific: DTDP induced no changes in expressed human cardiac Na current. Single-channel Ba2+ current recordings revealed a reduction in open probability and mean open time by DTDP but no change in single -channel conductance, implying that the reduction of macroscopic Ca2current reflects changes in gating and not permeation. In summary, the pore-forming (alpha(1)) subunit of the L-type Ca2+ channel contains f unctionally important free sulfhydryl groups that modulate gating. The se free sulfhydryl groups are accessible from the extracellular side b y an aqueous pathway.