Hongjian Xu

The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4+ T cells

TRPV1 is a Ca2+-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4+ T cells, where it acted as a non–store-operated Ca2+ channel and contributed to T cell antigen receptor (TCR)-induced Ca2+ influx, TCR signaling and T cell activation. In models of T cell–mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4+ T cells recapitulated the phenotype of mouse Trpv1−/− CD4+ T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

The molecular basis of high-affinity binding of the antiarrhythmic compound vernakalant (RSD1235) to Kv1.5 channels.

Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial fibrillation rapidly and safely in patients (J Am Coll Cardiol 44:2355–2361, 2004). Here, the molecular mechanisms of interaction of vernakalant with the inner pore of the Kv1.5 channel are compared with those of the class IC agent flecainide. Initial experiments showed that vernakalant blocks activated channels and vacates the inner vestibule as the channel closes, and thus mutations were made, targeting residues at the base of the selectivity filter and in S6, by drawing on studies of other Kv1.5-selective blocking agents. Block by vernakalant or flecainide of Kv1.5 wild type and mutants was assessed by whole-cell patch-clamp experiments in transiently transfected human embryonic kidney 293 cells. The mutational scan identified several highly conserved amino acids, Thr479, Thr480, Ile502, Val505, and Val508, as important residues for affecting block by both compounds. In general, mutations in S6 increased the IC50 for block by vernakalant; I502A caused an extremely local 25-fold decrease in potency. Specific changes in the voltage-dependence of block with I502A supported the crucial role of this position. A homology model of the pore region of Kv1.5 predicted that, of these residues, only Thr479, Thr480, Val505, and Val508 are potentially accessible for direct interaction, and that mutation at additional sites studied may therefore affect block through allosteric mechanisms. For some of the mutations, the direction of changes in IC50 were opposite for vernakalant and flecainide, highlighting differences in the forces that drive drug-channel interactions.

Internalized Kv1.5 traffics via Rab-dependent pathways

Little is known about the postinternalization trafficking of surface‐expressed voltage‐gated potassium channels. Here, for the first time, we investigate into which of four major trafficking pathways a voltage‐gated potassium channel is targeted after internalization. In both a cardiac myoblast cell line and in HEK293 cells, channels were found to internalize and to recycle quickly. Upon internalization, Kv1.5 rapidly associated with Rab5‐and Rab4‐positive endosomes, suggesting that the channel is internalized via a Rab5‐dependent pathway and rapidly targeted for recycling to the plasma membrane. Nevertheless, as indicated by colocalization with Rab7, a fraction of the channels are targeted for degradation. Recycling through perinuclear endosomes is limited; colocalization with Rab11 was evident only after 24 h postsurface labelling. Expression of dominant negative (DN) Rab constructs significantly increased Kv1.5 functional expression. In the myoblast line, Rab5DN increased Kv1.5 current densities to 1305 ± 213 pA pF−1 from control 675 ± 81.6 pA pF−1. Rab4DN similarly increased Kv1.5 currents to 1382 ± 155 pA pF−1 from the control 522 ± 82.7 pA pF−1 at +80 mV. Expression of the Rab7DN increased Kv1.5 currents 2.5‐fold in HEK293 cells but had no significant effect in H9c2 myoblasts, and, unlike the other Rab GTPases tested, over‐expression of wild‐type Rab7 decreased Kv1.5 currents in the myoblast line. Densities fell to 573 ± 96.3 pA pF−1 from the control 869 ± 135.5 pA pF−1. The Rab11DN was slow to affect Kv1.5 currents but had comparable effects to other dominant negative constructs after 48 h. With the exception of Rab11DN and nocodazole, the effects of interference with microtubule‐dependent trafficking by nocodazole or p50 overexpression were not additive with the Rab dominant negatives. The Rab GTPases thus constitute dynamic targets by which cells may modulate Kv1.5 functional expression.

Kif5b is an essential forward trafficking motor for the Kv1.5 cardiac potassium channel

We have investigated the role of the kinesin I isoform Kif5b in the trafficking of a cardiac voltage‐gated potassium channel, Kv1.5. In Kv1.5‐expressing HEK293 cells and H9c2 cardiomyoblasts, current densities were increased from control levels of 389 ± 50.0 and 317 ± 50.3 pA pF−1, respectively, to 614 ± 74.3 and 580 ± 90.9 pA pF−1 in cells overexpressing the Kif5b motor. Overexpression of the Kif5b motor increased Kv1.5 expression additively with several manipulations that reduce channel internalization, suggesting that it is involved in the delivery of the channel to the cell surface. In contrast, expression of a Kif5b dominant negative (Kif5bDN) construct increased Kv1.5 expression non‐additively with these manipulations. Thus, the dominant negative acts by indirectly inhibiting endocytosis. The increase in Kv1.5 currents induced by wild‐type Kif5b was dependent on Golgi function; a 6 h treatment with Brefeldin A reduced Kv1.5 currents to control levels in Kif5b‐overexpressing cells but had little effect on the increase associated with Kif5bDN expression. Finally, expression of the Kif5bDN prior to induction of Kv1.5 in a tetracycline inducible system blocked surface expression of the channel in both HEK293 cells and H9c2 cardiomyoblasts. Thus, Kif5b is essential to anterograde trafficking of a cardiac voltage‐gated potassium channel.

Mechanistic basis for LQT1 caused by S3 mutations in the KCNQ1 subunit of IKs

Long QT interval syndrome (LQTS) type 1 (LQT1) has been reported to arise from mutations in the S3 domain of KCNQ1, but none of the seven S3 mutations in the literature have been characterized with respect to trafficking or biophysical deficiencies. Surface channel expression was studied using a proteinase K assay for KCNQ1 D202H/N, I204F/M, V205M, S209F, and V215M coexpressed with KCNE1 in mammalian cells. In each case, the majority of synthesized channel was found at the surface, but mutant IKs current density at +100 mV was reduced significantly for S209F, which showed ∼75% reduction over wild type (WT). All mutants except S209F showed positively shifted V1/2’s of activation and slowed channel activation compared with WT (V1/2 = +17.7 ± 2.4 mV and τactivation of 729 ms at +20 mV; n = 18). Deactivation was also accelerated in all mutants versus WT (126 ± 8 ms at −50 mV; n = 27), and these changes led to marked loss of repolarizing currents during action potential clamps at 2 and 4 Hz, except again S209F. KCNQ1 models localize these naturally occurring S3 mutants to the surface of the helices facing the other voltage sensor transmembrane domains and highlight inter-residue interactions involved in activation gating. V207M, currently classified as a polymorphism and facing lipid in the model, was indistinguishable from WT IKs. We conclude that S3 mutants of KCNQ1 cause LQTS predominantly through biophysical effects on the gating of IKs, but some mutants also show protein stability/trafficking defects, which explains why the kinetic gain-of-function mutation S209F causes LQT1.

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG+) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs+, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K+, suggesting that K+ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG+ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K+, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.

Structure-Based Virtual Screening and Electrophysiological Evaluation of New Chemotypes of Kv1.5 Channel Blockers

Atrial fibrillation (AF) is the most prevalent nonfatal cardiac rhythm disorder associated with an increased risk of heart failure and stroke. Considering the ventricular side effects induced by anti‐arrhythmic agents in current use, Kv1.5 channel blockers have attracted a great deal of deliberation owing to their selective actions on atrial electrophysiology. Herein we report new chemotypes of Kv1.5 channel blockers that were identified through a combination of structure‐based virtual screening and in silico druglike property prediction including six scoring functions, as well as electrophysiological evaluation. Among them, five of the 18 compounds exhibited >50 % blockade ratio at 10 μM, and have structural features different from conventional Kv1.5 channel blockers. These novel scaffolds could serve as hits for further optimization and SAR studies for the discovery of selective agents to treat AF.

Voltage Sensor Immobilization in Kv1.2 Channels

The voltage gated potassium channel family contains several functionally distinct isoforms which serve to shape the duration, frequency and timing of action potential firing in electrically excitable cells. Several mechanistic elements that contribute to the function of the voltage sensing apparatus are not yet fully described, especially in channels other than the prototypical Shaker potassium channel. We have studied voltage sensor rearrangements of Kv1.2 channels using two-electrode voltage clamp fluorometry and gating current recordings in mammalian cells. Fluorescence measurements reporting on voltage sensor movement revealed a transition into a relaxed state upon prolonged depolarization, causing a left shift in the fluorescence-voltage relationship. Gating current measurements of wild type Kv1.2 channels recorded in permeant ion free solutions (NMDG+int//TEA+ext) also displayed a left shifted Q-V after depolarizing pre-pulses, reflecting a stabilization of the activated state of the voltage sensor. To enable examination of the effects of different cations and processes of inactivation in Kv1.2 a non-conducting double mutant channel mimicking the permanently slow inactivated and non-conducting Shaker W434F, Kv1.2(W366F, V381T) was created. Off-gating currents recorded from this channel in the presence of permeant ions displayed less voltage sensor stabilization in the activated state than the wild type channel. These data suggest that cations play a specific role in regulating voltage sensor dynamics in the Kv1.2 channel.