Functional Consequences of Sulfhydryl Modification in the Pore-Forming Subunits of Cardiovascular Ca ²⁺ and Na ⁺ Channels

Abstract

The structure and function of many cysteine-containing proteins critically depend on the oxidation state of the sulfhydryl groups. In such proteins, selective modification of sulfhydryl groups can be used to probe the relation between structure and function. We examined the effects of sulfhydryloxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit smooth muscle L-type Ca2+ channel and the human cardiac tetrodotoxin-insensitive 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 cysteines in regions of unknown function. We determined the effects of sulfhydryl modification on Ca2+ and Na+ channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp technique. Within 10 minutes of exposure to 2,2'-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca2+ current was reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The effect could be readily reversed by 1,4-dithiothreitol (an agent that reduces disulfide bonds). Similar results were obtained by using the hydrophilic sulfhydryl-oxidizing agent thimerosal. The effects were Ca(2+)-channel 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 Ca2+ current reflects changes in gating and not permeation. In summary, the pore-forming (alpha 1) subunit of the L-type Ca2+ channel contains functionally important free sulfhydryl groups that modulate gating. These free sulfhydryl groups are accessible from the extracellular side by an aqueous pathway.

NADPH Oxidase Contributes to Angiotensin II Signaling in the Nucleus Tractus Solitarius

“…This finding is consistent with the report that ROS, especially superoxide, enhance Ca 2ϩ currents only at negative depolarizing voltages (Li et al, 1998;Annunziato et al, 2002); however, the mechanisms by which ROS alter Ca 2ϩ channel activities remain poorly understood (Kourie, 1998). One possibility is that ROS produce oxidation of sulfhydryl groups of cystine residues, resulting in the formation of disulfide bonds (S-S) that modify the structure-function relationship of Ca 2ϩ channel proteins (Chiamvimonvat et al, 1995;Kourie, 1998;Li et al, 1998). Another possibility is that ROS lead to peroxidation of membrane phospholipids, altering the structure of the channel proteins and their ionic conductance (Kourie, 1998).…”

Redox modulation of basal and β‐adrenergically stimulated cardiac L‐type Ca2+ channel activity by phenylarsine oxide

“…This provides many potential targets for redox regulation of the channel protein, and redox modulation of the basal L-type Ca 2 þ current has been reported by several groups (Chiamvimonvat et al, 1995;Lacampagne et al, 1995;Campbell et al, 1996;Fearon et al, 1999;Yamaoka et al, 2000). However, there is no unifying explanation for the diversity of results that have been obtained.…”

“…Therefore, it is conceivable that PAO might affect L-type Ca 2 þ channel function by redox mechanisms that do not involve the inhibition of tyrosine phosphatase activity. In fact, oxidizing and reducing agents have been reported to have varied effects on the basal L-type Ca 2 þ current in cardiac myocytes (Chiamvimonvat et al, 1995;Lacampagne et al, 1995;Campbell et al, 1996;Fearon et al, 1999;Yamaoka et al, 2000). However, the effect that changes in the redox state of the cell might have on b-adrenergic regulation of the L-type Ca 2 þ current have not been thoroughly investigated.…”

Oxidoreductase regulation of Kv currents in rat ventricle

“…Many cardiac ion channels have been shown to be sensitive to ROS and related thiol-reactive molecules in a reversible manner that suggests the channel protein itself or a modulator of the channel is controlled by cellular redox state [14][15][16][17]. In most cases, the reversibility or prevention of oxidative changes to ion channels has been examined using exogenous reductants such as dithiothreitol.…”