Dennis Wray

Functional and molecular aspects of voltage-gated K+ channel beta subunits

ABSTRACT: Voltage‐gated potassium channels (Kv) of the Shaker‐related superfamily are assembled from membrane‐integrated α subunits and auxiliary β subunits. The β subunits may increase Kv channel surface expression and/or confer A‐type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kvα and Kvβ subunits depends on the presence or absence of several domains including the amino‐terminal N‐type inactivating and NIP domains and the Kvα and Kvβ binding domains. Loss of function of Kvβ1.1 subunits leads to a reduction of A‐type Kv channel activity in hippocampal and striatal neurons of knock‐out mice. This reduction may be correlated with altered cognition and motor control in the knock‐out mice.

MEASUREMENT OF THE MOVEMENT OF THE S4 SEGMENT DURING THE ACTIVATION OF A VOLTAGE-GATED POTASSIUM CHANNEL

Abstract Voltage-gated ion channels contain a positively charged transmembrane segment termed S4. Recent evidence suggests that depolarisation of the membrane potential causes this segment to undergo conformational changes that, in turn, lead to the opening of the channel pore. In order to define these conformational changes in structural terms, we have introduced single cysteine substitutions into the S4 segment of the prototypical Shaker K+ channel at various positions and expressed the mutants in Xenopus oocytes. The cells were depolarised to induce K+ currents and the effect of application of 100 μM parachloromercuribenzenesulphonate (PCMBS) on these currents was examined by the two-electrode voltage-clamp technique. PCMBS inhibited K+ currents elicited by mutants L358C, L361C, V363C and L366C, but not those by V367C and S376C. Since PCMBS is a membrane-impermeable cysteine-modifying reagent, the data suggest that depolarisation must have caused the S4 segment to move out of the lipid bilayer into the extracellular phase rendering the residues at positions 358, 361, 363 and 366 susceptible to PCMBS attack. The lack of effect of PCMBS on V367C suggests that the exposure of S4 terminates at L366. Detailed analysis of L361C mutant revealed that the S4 movement can occur even below the resting potential of the cell, at which potential voltage-gated K+ channels are normally in a non-conducting closed state.

Molecular identification and characterisation of the human eag2 potassium channel

We report the molecular cloning from foetal brain of the human potassium channel heag2. The cDNA encodes a protein of 988 amino acids, 73% identical to heag1. Heag2 is expressed in the brain, but is also found in a range of tissues including skeletal muscle. In oocytes, the channel is a non‐inactivating outward rectifier, with dependence of activation rate on holding potential. Compared with heag1, the conductance–voltage curve for heag2 was shifted to the left, the voltage sensitivity was less, activation kinetics were different, and the sensitivity to terfenadine was lower. The heag2 channel may have important physiological roles.

Use of site-directed antibodies to probe the topography of the alpha 2 subunit of voltage-gated Ca2+ channels.

Polyclonal antibodies were raised against peptides corresponding to residues 1–15, 469–483 and 933–951 of the rabbit skeletal muscle L‐type calcium channelα2/δ primary translation product, for use as topological probes. Immunocytochemical comparison of the abilities of the antibodies to bind to theα2 and δ subunits in intact and detergent‐permeabilised rat dorsal root ganglion cells enabled the membrane orientation of these regions to be established. The resultant data indicate that the regions containing residues 1–15 and 469–483 of theα2 subunit, and residues 1–17 of the δ subunit, are exposed on the extracellular surface of the membrane, findings consistent with a model that proposesα2 to be entirely extracellular.

Role of intracellular domains in the function of the herg potassium channel

The functional role of the large intracellular regions (which include the cyclic nucleotide binding domain, cNBD, and the Per-Arnt-Sim domain, PAS) in the herg channel is not well understood. We have studied possible interactions of the cNBD with other parts of the channel protein using lysine mutations to disrupt such interactions. Some lysine mutations caused significant right shifts in the voltage dependence of inactivation; almost all the mutants caused speeding up of deactivation time course. In a homology model of the cNBD, lysine mutations that affected both inactivation and deactivation lie in a hydrophobic band on the surface of the structure of this domain. Some known mutations in the Long QT Syndrome type 2, with effects on deactivation, are located at residues close to hydrophobic bands on the cNBD and the PAS domains. Such bands of residues in these intracellular domains may play an important part in channel function.

Modulation by protein kinase A of a cloned rat brain potassium channel expressed in Xenopus oocytes

A potassium channel from rat brain was expressed in Xenopus oocytes in order to study modulation of channel function by phosphorylation via protein kinase A. Application of 8-Br-cAMP to oocytes expressing the drk1 channel (with the first 139 amino acids of the N terminus delected, ΔNdrk1) caused a voltage-independent elevation of current amplitude, which was not seen for endogenous currents or for wild-type full-length drk1 channel. This effect on ΔNdrk1 was blocked by pre-injection of oocytes with Walsh-peptide protein kinase A inhibitor, suggesting mediation via protein kinase A. The protein kinase inhibitor also reduced both ΔNdrk1 and full-length drk1 currents. Substitution of the serine residues by alanine at one or both of the two consensus protein kinase A phosphorylation sites on the C terminus (residues 440 and 492) of ΔNdrk1 resulted in a loss of function of the expressed channels. These results indicate that phosphorylation via protein kinase A modulates drk1 channel function and that both consensus phosphorylation sites seems to be essential for channels to function.

Effect of cysteine substitutions on the topology of the S4 segment of the Shaker potassium channel: implications for molecular models of gating

1 The gating properties of voltage‐gated potassium channels are largely determined by the amino acid sequence of their S4 segments. To investigate the nature of S4 movement during gating, we introduced single cysteines into the S4 segment of the Shaker potassium channel and expressed the mutants in Xenopus oocytes. We then measured the conductance‐voltage (g‐V) relationships and the rate and the voltage dependence of movement of the engineered cysteines, using p‐chloromercuribenzene sulphonate (pCMBS) as a probe. 2 Mutation of charged residues at positions 362, 365 and 368, but not the uncharged residues (positions 360, 361, 363, 364 and 366), to cysteines shifted the g‐V relationships to more positive potentials. Mutant channels in which cysteines replaced the charged residues at positions 362 and 365 (R362C and R365C) reacted faster with pCMBS than those in which cysteines were introduced in place of uncharged residues at positions 360 and 361 (I360C and L361C). Furthermore, the R365C mutant channel reacted with pCMBS even at hyperpolarised (‐120 mV) potentials. Currents expressed by the doubly mutated R365S/V367C and R368S/V367C channels, but not the singly mutated V367C channel, were inhibited by pCMBS. Moreover, the R368C mutant channel was also affected by pCMBS. 3 Voltage dependence of block by pCMBS (2 min exposure) was steeper for L366C than for L361C and V363C mutant channels (effective charge 2.19, 1.41 and 1.45, respectively). The voltage dependence of the pCMBS effect was also shifted to more depolarising potentials the deeper in the membrane the position of the residue mutated to cysteine (voltages for half‐maximal effect ‐107, ‐94 and ‐73 mV for positions 361, 363 and 366, respectively). 4 Our data show firstly that charge‐neutralising mutations in S4 alter the topology of this region such that the membrane‐spanning portion of S4 is reduced. Secondly, our data for the other mutant channels suggest that S4 might move in at least two sequential steps, and can move up to its maximal limit even at the resting potential of the cell.

Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating

The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFPN-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.

Structural determinants of drugs acting on the Nav1.8 channel.

The aim of this work is to study the role of pore residues on drug binding in the NaV1.8 channel. Alanine mutations were made in the S6 segments, chosen on the basis of their roles in other NaV subtypes; whole cell patch clamp recordings were made from mammalian ND7/23 cells. Mutations of some residues caused shifts in voltage dependence of activation and inactivation, and gave faster time course of inactivation, indicating that the residues mutated play important roles in both activation and inactivation in the NaV1.8 channel. The resting and inactivated state affinities of tetracaine for the channel were reduced by mutations I381A, F1710A, and Y1717A (for the latter only inactivated state affinity was measured), and by mutation F1710A for the NaV1.8-selective compound A-803467, showing the involvement of these residues for each compound, respectively. For both compounds, mutation L1410A caused the unexpected appearance of a complete resting block even at extremely low concentrations. Resting block of native channels by compound A-803467 could be partially removed (“disinhibition”) by repetitive stimulation or by a test pulse after recovery from inactivation; the magnitude of the latter effect was increased for all the mutants studied. Tetracaine did not show this effect for native channels, but disinhibition was seen particularly for mutants L1410A and F1710A. The data suggest differing, but partially overlapping, areas of binding of A-803467 and tetracaine. Docking of the ligands into a three-dimensional model of the NaV1.8 channel gave interesting insight as to how the ligands may interact with pore residues.

The roles of intracellular regions in the activation of voltage-dependent potassium channels

The involvement of the transmembrane regions S2, S3 and S4 in the activation of potassium channels by depolarization has been well clarified. However, a role of the intracellular regions in channel function is emerging. Here we review recent evidence for the roles of intracellular regions in the functioning of members of two families of channels. The Kv2.1 potassium channel, a member of the voltage activated Kv family, has long intracellular regions. By mutagenesis studies and expression in oocytes, we identify residues in both the N- and C-terminal regions that contribute to determining activation kinetics of this channel. It seems that the C-terminus wraps around the N-terminus and interacts with it functionally. The voltage-activated ether-a-go-go (eag) channels also have long intracellular regions. Despite considerable homology, eag1 and eag2 channels display different activation kinetics. By making chimeras between these channels and again expressing in oocytes, we show that residues in both the N-terminal region and the membrane-spanning region are involved in determining these differences in activation kinetics. The intracellular N- and C-terminal regions are likely to continue to prove fertile regions in future investigations into the functioning of ion channels.