C-type inactivation in the HERG channel is unique among voltage-gated K channels in having extremely fast kinetics and strong voltage sensitivity. This suggests that HERG may have a unique outer mouth structure (where conformational changes underlie C-type inactivation), and/or a unique communication between the outer mouth and the voltage sensor. We use cysteine-scanning mutagenesis and thiol-modifying reagents to probe the structural and functional role of the S5-P (residues 571–613) and P-S6 (residues 631–638) linkers of HERG that line the outer vestibule of the channel. Disulfide formation involving introduced cysteine side chains or modification of side chain properties at “high-impact” positions produces a common mutant phenotype: disruption of C-type inactivation, reduction of K+ selectivity, and hyperpolarizing shift in the voltage-dependence of activation. In particular, we identify 15 consecutive positions in the middle of the S5-P linker (583–597) where side chain modification has marked impact on channel function. Analysis of the degrees of mutation-induced perturbation in channel function along 583–597 reveals an α-helical periodicity. Furthermore, the effects of MTS modification suggest that the NH2-terminal of this segment (position 584) may be very close to the pore entrance. We propose a structural model for the outer vestibule of the HERG channel, in which the 583–597 segment forms an α-helix. With the NH2 terminus of this helix sitting at the edge of the pore entrance, the length of the helix (∼20 Å) allows its other end to reach and interact with the voltage-sensing domain. Therefore, the “583–597 helix” in the S5-P linker of the HERG channel serves as a bridge of communication between the outer mouth and the voltage sensor, that may make important contribution to the unique C-type inactivation phenotype.

The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-gorelated gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteinescanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx⅐Shaker interaction, elevating [K] o (from 2 to 98 mM) does not affect ErgTx potency, and throughsolution electrostatic forces only play a minor role in influencing ErgTx⅐HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (⌬⌬G > 2 kcal/ mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic ␣-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx⅐HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.

KCNQ1 and KCNE1 (Q1 and E1) associate to form the slow delayed rectifi er I Ks channels in the heart. A short stretch of eight amino acids at the extracellular end of S1 in Q1 (positions 140 -147) harbors six arrhythmia-associated mutations. Some of these mutations affect the Q1 channel function only when coexpressed with E1, suggesting that this Q1 region may engage in the interaction with E1 critical for the I Ks channel function. Identifying the Q1/E1 contact points here may provide new insights into how the I Ks channel operates. We focus on Q1 position 145 and E1 positions 40 -43. Replacing all native cysteine (Cys) in Q1 and introducing Cys into the above Q1 and E1 positions do not signifi cantly perturb the Q1 channel function or Q1/E1 interactions. Immunoblot experiments on COS-7 cells reveal that Q1 145C can form disulfi de bonds with E1 40C and 41C, but not E1 42C or 43C. Correspondingly, voltage clamp experiments in oocytes reveal that Q1 145C coexpressed with E1 40C or E1 41C manifests unique gating behavior and DTT sensitivity. Our data suggest that E1 40C and 41C come close to Q1 145C in the activated and resting states, respectively, to allow disulfi de bond formation. These data and those in the literature lead us to propose a structural model for the Q1/E1 channel complex, in which E1 is located between S1, S4, and S6 of three separate Q1 subunits. We propose that E1 is not a passive partner of the Q1 channel, but instead can engage in molecular motions during I Ks gating.on May 7, 2018 jgp.rupress.org Downloaded from

We have identified the tRNAs which are incorporated into both wild-type human immunodeficiency virus type 1 strain HIB (HIV-111,B) produced in COS-7 cells transfected with HlV-1 proviral DNA and mutant, noninfectious HIV-1L, particles produced in a genetically engineered Vero cell line. The mutant proviral DNA contains nucleotides 678 to 8944; i.e., both long terminal repeats and the primer binding site are absent. As analyzed by two-dimensional polyacrylamide gel electrophoresis, both mutant and wild-type HIV-1 contain four major-abundance tRNA species, which include tRNALYS, tRNAI"Y (the putative primer for HIV-1 reverse transcriptase) and tRNA". Identification was accomplished by comparing the electrophoretic mobilities and RNase T, digests with those of tRNA3LY' and tRNAj', purified from human placenta and comparing the partial nucleotide sequence at the 3' end of each viral tRNA species with published tRNA sequences. Thus, the absence of the primer binding site in the mutant virus does not affect tRNALYS incorporation into HIV-1. However, only the wild-type virus contains tRNALY" tightly associated with the viral RNA genome. The identification of the * Corresponding author.

COS-7 cells transfected with human immunodeficiency virus type 1 (HIV-1) proviral DNA produce virus in which three tRNA species are most abundant in the viral tRNA population. These tRNAs have been identified through RNA sequencing techniques as tRNA3LY the primer tRNA in HIV-1, and members of the tRNALYS isoacceptor family. These RNAs represent 60% of the low-molecular-weight RNA isolated from virus particles, while they represent only 6% of the low-molecular-weight RNA isolated from the COS cell cytoplasm. Thus,

The transmembrane domains of HERG (S1–S3) contain six negative charges: three are conserved in all voltage-gated K channels (D456 and D466 in S2, D501 in S3) and three are unique to the EAG family (D411 in S1, D460 in S2, and D509 in S3). We infer the functional role of these aspartates by studying how substituting them with cysteine, one at a time, affects the channel function. D456C is not functional, suggesting that this negative charge may play a critical role in channel protein folding during biogenesis, as has been shown for its counterpart in the Shaker channel. Data from the other five functional mutants suggest that D411 can stabilize the HERG channel in the closed state, while D460 and D509 have the opposite effect. D466 and D501 both may contribute to voltage-sensing during the activation process. On the other hand, all five aspartates work in a concerted fashion in contributing to the slow deactivation process of the HERG channel. Accessibility tests of the introduced thiol groups to extracellular MTS reagents indicate that water-filled crevices penetrate deep into the HERG protein core, reaching the cytoplasmic halves of S1 and S2. At these deep locations, accessibility of 411C and 466C to the extracellular aqueous phase is voltage dependent, suggesting that conformational changes occur in S1 and S2 or the surrounding crevices during gating. Increasing extracellular [H+] accelerates HERG deactivation. This effect is suppressed by substituting the aspartates with cysteine, suggesting that protonation of these aspartates may contribute to the signaling pathway whereby external [H+] influences conformational changes in the channel's cytoplasmic domains (where deactivation takes place). There is no evidence for a metal ion binding site coordinated by negative charges in the transmembrane domains of HERG, as the one described for the EAG channel.

Background-Mutations in KCNE2 have been linked to long-QT syndrome (LQT6), yet KCNE2 protein expression in the ventricle and its functional role in native channels are not clear. Methods and Results-We detected KCNE2 protein in human, dog, and rat ventricles in Western blot experiments.Immunocytochemistry confirmed KCNE2 protein expression in ventricular myocytes. To explore the functional role of KCNE2, we studied how its expression was altered in 2 models of cardiac pathology and whether these alterations could help explain observed changes in the function of native channels, for which KCNE2 is a putative auxiliary (␤) subunit. In canine ventricle injured by coronary microembolizations, the rapid delayed rectifier current (I Kr ) density was increased. Although the protein level of ERG (I Kr pore-forming, ␣, subunit) was not altered, the KCNE2 protein level was markedly reduced. These data are consistent with the effect of heterologously expressed KCNE2 on ERG and suggest that in canine ventricle, KCNE2 may associate with ERG and suppress its current amplitude. In aging rat ventricle, the pacemaker current (I f ) density was increased. There was a significant increase in the KCNE2 protein level, whereas changes in the ␣-subunit (HCN2) were not significant. These data are consistent with the effect of heterologously expressed KCNE2 on HCN2 and suggest that in aging rat ventricle, KCNE2 may associate with HCN2 and enhance its current amplitude. Conclusions-KCNE2 protein is expressed in ventricles, and it can play diverse roles in ventricular electrical activity under (patho)physiological conditions. (Circulation. 2004;109:1783-1788.)Key Words: ion channels Ⅲ electrophysiology Ⅲ hypertrophy T he KCNE gene family encodes small proteins (103 to 170 amino acids) with 1 transmembrane domain. 1 They function as ␤-subunits of voltage-gated cation channels by associating with ␣-subunits and modulating their properties. [2][3][4][5][6][7] Heterologously expressed KCNE1-KCNE3 can interact with multiple target ␣-subunits. 2-5,8 -11 Conversely, some ␣-subunits can interact with multiple KCNE subunits. 2,6,7,9,11 Therefore, the relationships between KCNE and ␣-subunits are complicated.Among the KCNE subunits, KCNE2 seems to be the most promiscuous one. Heterologously expressed KCNE2 can associate with ERG (I Kr ␣-subunit), 3 Kv4.x (xϭ2 or 3) 4 and Kv3.4 (M. Pourrier, PhD, et al, unpublished data, 2002) (transient outward or I to ␣-subunit), KCNQ1 (slow delayed or I Ks ␣-subunit 9 ; M. Zhang, PhD, et al, unpublished data, 2003), and HCNx (xϭ1 or 2, I f ␣-subunits). 10 The importance of KCNE2 in maintaining the ventricular electrical stability is suggested by the linkage between inherited mutations or a polymorphism in KCNE2 and sporadic or acquired long-QT syndrome (LQT6). 3,12 Investigators studying the role of KCNE2 in ventricular repolarization and arrhythmogenic mechanism(s) of LQT6 face two problems. First, expression of KCNE2 protein in the ventricle has not been established. 13 Second, so far, all the experiments s...

Human ether-à-go-go-related gene (HERG) encodes a K channel similar to the rapid delayed rectifier channel current (I(Kr)) in cardiac myocytes. Modulation of I(Kr) by extracellular acidosis under pathological conditions may impact on cardiac electrical activity. Therefore, we studied the effects of extracellular acidification on I(Kr) function and the underlying mechanism, using HERG expressed in Xenopus oocytes as a model. Acidification [extracellular pH (pH(o)) 8.5-6.5] accelerated HERG deactivation (at -80 mV, the time constant tau of the major component of deactivation was 253 +/- 17, 158 +/- 10, and 65 +/- 5 ms at pH(o) 8.5, 7.5, and 6.5, respectively; n = 7-10 each), with no effects on other gating kinetics except a modest acceleration of recovery from inactivation (at -80 mV, tau of recovery was 4.7 +/- 0.3, 3.8 +/- 0.3, and 1.3 +/- 0.2 ms at pH(o) 8. 5, 7.5, and 6.5, respectively; n = 4-7 each). The following were ruled out as the underlying mechanisms: 1) voltage shift in channel activation, 2) pore blockade by protons, 3) protonation of histidines on the extracellular domain of HERG, 4) acceleration of recovery from C-type inactivation, and 5) interaction between an external H(+) binding site and the cytoplasmic NH(2)-terminal domain (a key determinant of HERG deactivation rate). Extracellular application of diethylpyrocarbonate caused an irreversible acceleration of HERG deactivation and prevented further acceleration by external acidification. Our data suggest that side chains accessible to the extracellular solution mediated the effects of elevating extracellular H(+) concentration on channel deactivation.