In response to membrane depolarization, voltage-gated ion channels undergo a structural rearrangement that moves charges or dipoles in the membrane electric field and opens the channel-conducting pathway. By combination of site-specific fluorescent labeling of the Shaker potassium channel protein with voltage clamping, this gating conformational change was measured in real time. During channel activation, a stretch of at least seven amino acids of the putative transmembrane segment S4 moved from a buried position into the extracellular environment. This movement correlated with the displacement of the gating charge, providing physical evidence in support of the hypothesis that S4 is the voltage sensor of voltage-gated ion channels.

We have acquired structural evidence that two components evident previously in the depolarization-evoked gating currents from voltage-gated Shaker K+ channels have their origin in sequential, two-step outward movements of the S4 protein segments. A point mutation greatly destabilizes the "fully retracted" state of S4 transmembrane translocation, causing instead an intermediate state to predominate at resting potentials. This state is distinguishable topologically and fluorometrically. That a point mutation effectively excludes half the range of S4 motion from physiological voltages suggests that the diverse sensitivities among voltage-gated channels might reflect not only differences in S4 valence, but also displacement. Existence of an intermediate subunit state helps explain why modeling channel activation has required positing greater than four closed states.

Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing it is necessary to define the structure and motion of the S4 segment, the portion of each voltage-sensing domain that moves charged residues across the membrane in response to voltage change. We have addressed this problem by using fluorescence resonance energy transfer as a spectroscopic ruler to determine distances between S4s in the Shaker K+ channel in different gating states. Here we provide evidence consistent with S4 being a tilted helix that twists during activation. We propose that helical twist contributes to the movement of charged side chains across the membrane electric field and that it is involved in coupling voltage sensing to gating.

Voltage-gated potassium channels are composed of four subunits. Voltage-dependent activation of these channels consists of a depolarization-triggered series of charge-carrying steps that occur in each subunit. These major charge-carrying steps are followed by cooperative step(s) that lead to channel opening. Unlike the late cooperative steps, the major charge-carrying steps have been proposed to occur independently in each of the channel subunits. In this paper, we examine this further. We showed earlier that the two major charge-carrying steps are associated with two sequential outward transmembrane movements of the charged S4 segment. We now use voltage clamp fluorometry to monitor these S4 movements in individual subunits of heterotetrameric channels. In this way, we estimate the influence of one subunit's S4 movement on another's when the energetics of their transmembrane movements differ. Our results show that the first S4 movement occurs independently in each subunit, while the second occurs cooperatively. At least part of the cooperativity appears to be intrinsic to the second S4 charge-carrying rearrangement. Such cooperativity in gating of voltage-dependent channels has great physiological relevance since it can affect both action potential threshold and rate of propagation.

Total internal reflection fluorescence microscopy was used to detect single fluorescently labeled voltage-gated Shaker K ؉ channels in the plasma membrane of living cells. Tetramethylrhodamine (TMR) attached to specific amino acid positions in the voltagesensing S4 segment changed fluorescence intensity in response to the voltage-driven protein motions of the channel. The voltage dependence of the fluorescence of single TMRs was similar to that seen in macroscopic epi-illumination microscopy, but the exclusion of nonchannel fluorescence revealed that the dimming of TMR upon voltage sensor rearrangement was much larger than previously thought, and is due to an extreme, Ϸ20-fold suppression of the elementary fluorescence. The total internal reflection voltageclamp method reveals protein motions that do not directly open or close the ion channel and which have therefore not been detected before at the single-channel level. The method should be applicable to a wide assortment of membrane-associated proteins and should make it possible to relate the structural rearrangements of single proteins to simultaneously measured physiological cellsignaling events. D ynamic readouts of the structural rearrangements in membrane proteins have been obtained from changes in the fluorescence of site-specifically attached dyes (1-11). In ion channels the fractional fluorescent change (⌬F) can be large and very specific, differing in direction and amplitude and in which functional step is detected as the dye-attachment site is moved from one residue to the next (2, 9, 10). The mechanism of the fluorescence report is not known.The fluorescence studies have so far been confined to large ensembles of proteins, where the variation from protein to protein in occupancy of distinct states blurs the transitions between them, even when activating signals are synchronized by voltage clamping. Such blurring can be avoided in singlemolecule determinations (12)(13)(14). Obstacles to optical detection have so far limited single-molecule optical studies of protein rearrangement to purified preparations, out of the native cellular context. We have overcome these obstacles for the optical detection of structural rearrangements in single-membrane proteins in living cells, enabling functional transitions that do not open or close gates to be detected on the single-channel level. Materials and Methods Molecular Biology.Fluorescence experiments were performed on nonconducting (W434F) (15), ball-deleted (⌬6-46) (16) ShH4 Shaker channels (17), after the removal of two native cysteines (C245V and C462A) (2) to ensure that membrane-impermeant fluorescent thiols would attach exclusively to a known position of cysteine addition. Site-directed mutagenesis, cRNA synthesis, and cRNA injection into Xenopus oocytes were as described (3), leading to high-density channel expression in all of the experiments.Oocyte Preparation. The vitelline membrane was found to be about 3 m thick by scanning electron microscopy (not shown) and to refract or scatter the excitation li...

Voltage-gated EAG K+ channels switch between fast and slow gating modes in a Mg2+-dependent manner by an unknown mechanism. We analyzed molecular motions in and around the voltage-sensing S4 in bEAG1. Using accessibility and perturbation analyses, we found that activation increases both the charge occupancy and volume of S4 side chains in the gating canal. Fluorescence measurements suggest that mode switching is due to a motion of the S2/S3 side of the gating canal. We propose that when S4 is in the resting state and its thin end is in the gating canal, a conformational rearrangement of S2/S3 narrows the canal around S4, forming the Mg2+ binding site. Binding of Mg2+ is proposed to stabilize this conformation and to slow opening of the gate by impeding S4's voltage-sensing outward motion.

In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that < or = 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2-6 x 10(6) sec-1 at 22 degrees C is calculated suggesting a channel mechanism.

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