Differential Asparagine-Linked Glycosylation of Voltage-Gated K+ Channels in Mammalian Brain and in Transfected Cells

Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked glycosylation of voltage-gated K⁺ channel α subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels, reflecting the different efficiencies of transport of K⁺ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich N-linked oligosaccharide chains differs among highly related K⁺ channel α subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential glycosylation contributing to diversity of K⁺ channel function in mammalian brain. Received: 17 December 1998/Accepted: 20 January 1999     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.     

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Δ40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K⁺. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Δ40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Δ40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.     

Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2Δ2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2Δ2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.     

The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

Regulation and Properties of KCNQ1 (KVLQT1) and Impact of the Cystic Fibrosis Transmembrane Conductance Regulator

The K⁺ channel KCNQ1 (K_VLQT1) is a voltage-gated K⁺ channel, coexpressed with regulatory subunits such as KCNE1 (IsK, mink) or KCNE3, depending on the tissue examined. Here, we investigate regulation and properties of human and rat KCNQ1 and the impact of regulators such as KCNE1 and KCNE3. Because the cystic fibrosis transmembrane conductance regulator (CFTR) has also been suggested to regulate KCNQ1 channels we studied the effects of CFTR on KCNQ1 in Xenopus oocytes. Expression of both human and rat KCNQ1 induced time dependent K⁺ currents that were sensitive to Ba²⁺ and 293B. Coexpression with KCNE1 delayed voltage activation, while coexpression with KCNE3 accelerated current activation. KCNQ1 currents were activated by an increase in intracellular cAMP, independent of coexpression with KCNE1 or KCNE3. cAMP dependent activation was abolished in N-terminal truncated hKCNQ1 but was still detectable after deletion of a single PKA phosphorylation motif. In the presence but not in the absence of KCNE1 or KCNE3, K⁺ currents were activated by the Ca²⁺ ionophore ionomycin. Coexpression of CFTR with either human or rat KCNQ1 had no impact on regulation of KCNQ1 K⁺ currents by cAMP but slightly shifted the concentration response curve for 293B. Thus, KCNQ1 expressed in Xenopus oocytes is regulated by cAMP and Ca²⁺ but is not affected by CFTR. Received: 13 December 2000/Revised: 30 March 2001     

Mutations in the M4 Domain of the Torpedo californica Nicotinic Acetylcholine Receptor Alter Channel Opening and Closing

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time. Received: 27 September 1996/Revised: 28 January 1997     

Role of the S2 and S3 Segment in Determining the Activation Kinetics in Kv2.1 Channels

We constructed chimeras between the rapidly activating Kv1.2 channel and the slowly activating Kv2.1 channel in order to study to what extent sequence differences within the S1–S4 region contribute to the difference in activation kinetics. The channels were expressed in Xenopus oocytes and the currents were measured with a two-microelectrode voltage-clamp technique. Substitution of the S1–S4 region of Kv2.1 subunits by the ones of Kv1.2 resulted in chimeric channels which activated more rapidly than Kv2.1. Furthermore, activation kinetics were nearly voltage-independent in contrast to the pronounced voltage-dependent activation kinetics of both parent channels. Systematic screening of the S1–S4 region by the replacement of smaller protein parts resolved that the main functional changes generated by the S1–S4 substitution were generated by the S2 and the S3 segment. However, the effects of these segments were different: The S3 substitution reduced the effective gating charge and accelerated both a voltage-dependent and a voltage-independent component of the activation time course. In contrast, the S2 substitution accelerated predominantly the voltage-dependent component of the activation time course thereby leaving the effective gating charge unchanged. It is concluded that the S2 and the S3 segment determine the activation kinetics in a specific manner. Received: 13 November 2000/Revised: 5 April 2001     

Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kvbeta subunits

The accessory beta subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.     

New Molecular Determinant for Inactivation of the Human L-Type α1C Ca2+ Channel

Molecular cloning of the human fibroblast Ca²⁺ channel pore-forming α_1C subunit revealed (Soldatov, 1992. Proc. Natl. Acad. Sci. USA 89:4628-4632) a naturally occurring mutation g²²⁵⁴→ a that causes the replacement of the conservative alanine for threonine at the position 752 at the cytoplasmic end of transmembrane segment IIS6. Using stably transfected HEK293 cell lines, we have compared electrophysiological properties of the conventional α_1C,77 human recombinant L-type Ca²⁺ channel with those of its mutated isoform α_1C,94 containing the A752T replacement. Comparative quantification of steady-state availability of the current carried by α_1C,94 and α_1C,77 showed that A752T mutation prevented a large (≈25%) fraction of the current carried by Ca²⁺ or Ba²⁺ from fully inactivating. This mutation, however, did not appear to alter significantly the Ca²⁺-dependence and kinetics of decay of the inactivating fraction of the current or its voltage-dependence. The data suggests that Ala752 at the cytoplasmic end of IIS6 might serve as a molecular determinant of the Ca²⁺ channel inactivation, possibly regulating the voltage-dependence of its availability. Received: 14 January 2000/Revised: 20 June 2000     

Native Kv1.3 Channels are Upregulated by Protein Kinase C

The voltage-gated potassium channel, Kv1.3, which is highly expressed in a number of immune cells, contains concensus sites for phosphorylation by protein kinase C (PKC). In lymphocytes, this channel is involved in proliferation—through effects on membrane potential, Ca²⁺ signalling, and interleukin-2 secretion—and in cytotoxic killing and volume regulation. Because PKC activation (as well as increased intracellular Ca²⁺) is required for T-cell proliferation, we have studied the regulation of Kv1.3 current by PKC in normal (nontransformed) human T lymphocytes. Adding intracellular ATP to support phosphorylation, shifted the voltage dependence of activation by +8 mV and inactivation by +17 mV, resulting in a 230% increase in the window current. Inhibiting ATP production and action with ``death brew' (2-deoxyglucose, adenylylimidodiphosphate, carbonyl cyanide-m-chlorophenyl hydrazone) reduced the K⁺ conductance (G _K ) by 41 ± 2%. PKC activation by 4β-phorbol 12,13-dibutyrate, increased G _K by 69 ± 6%, and caused a positive shift in activation (+9 mV) and inactivation (+9 mV), which resulted in a 270% increase in window current. Conversely, several PKC inhibitors reduced the current. Diffusion into the cell of inhibitory pseudosubstrate or substrate peptides reduced G _K by 43 ± 5% and 38 ± 8%, respectively. The specific PKC inhibitor, calphostin C, potently inhibited Kv1.3 current in a dose- and light-dependent manner (IC₅₀∼ 250 nm). We conclude that phosphorylation by PKC upregulates Kv1.3 channel activity in human lymphocytes and, as a result of shifts in voltage dependence, this enhancement is especially prevalent at physiologically relevant membrane potentials. This increased Kv1.3 current may help maintain a negative membrane potential and a high driving force for Ca²⁺ entry in the presence of activating stimuli. Received: 12 July 1996/Revised: 21 October 1996     

KCNE1 and KCNE2 inhibit forward trafficking of homomeric N-type voltage-gated potassium channels

Potassium currents generated by voltage-gated potassium (Kv) channels comprising α-subunits from the Kv1, 2, and 3 subfamilies facilitate high-frequency firing of mammalian neurons. Within these subfamilies, only three α-subunits (Kv1.4, Kv3.3, and Kv3.4) generate currents that decay rapidly in the open state because an N-terminal ball domain blocks the channel pore after activation—a process termed N-type inactivation. Despite its importance to shaping cellular excitability, little is known of the processes regulating surface expression of N-type α-subunits, versus their slowly inactivating (delayed rectifier) counterparts. Here we found that currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels are all strongly suppressed by the single transmembrane domain ancillary (β) subunits KCNE1 and KCNE2. A combination of electrophysiological, biochemical, and immunofluorescence analyses revealed this suppression is due to KCNE1 and KCNE2 retaining Kv1.4 and Kv3.4 intracellularly, early in the secretory pathway. The retention is specific, requires α-β coassembly, and does not involve the dynamin-dependent endocytosis pathway. However, the small fraction of Kv3.4 that escapes KCNE-dependent retention is regulated by dynamin-dependent endocytosis. The findings illustrate two contrasting mechanisms controlling surface expression of N-type Kv α-subunits and therefore, potentially, cellular excitability and refractory periods.     

KCNE1 and KCNE2 provide a checkpoint governing voltage-gated potassium channel α-subunit composition

Voltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and—by extension—cellular excitability.     

Sea Anemone Toxin (ATX II) Modulation of Heart and Skeletal Muscle Sodium Channel α-Subunits Expressed in tsA201 Cells

We have expressed recombinant α-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, τ^fast _h ) and toxin-bound (slow component, τ^slow _h ) channels. The relative amplitude of τ^slow _h allows an estimate of the IC₅₀ values ∼11 nm. The slowing of inactivation in the presence of ATX II is consistent with destabilization of the inactivated state by toxin binding. Further evidence for this conclusion is: (i) The voltage-dependence of the current decay time constants (τ _h ) is lost or possibly reversed (time constants plateau or increase at more positive voltages in contrast to these of untreated channels). (ii) The single channel mean open times are increased by a factor of two in the presence of ATX II. (iii) The recovery from inactivation is faster in the presence of ATX II. Similar effects of ATX II on rSkM1 channel behavior occur, but only at higher concentrations of toxin (IC₅₀= 51 nm). The slowing of inactivation on hSkM1 is comparable to the one seen with rSkM1. A residual or window current appears in the presence of ATX II that is similar to that observed in channels containing mutations associated with some of the familial periodic paralyses. Received: 5 December 1995/Revised: 1 March 1996     

Separate Domains for Desensitization of GABA ρ1 and β2 Subunits Expressed in Xenopus Oocytes

Desensitization of ligand-gated receptor channels is an intrinsic feedback mechanism and prevents the receptor/channels from becoming overly activated thereby maintaining biological function of the nervous system. Desensitization also plays an important role in neuronal plasticity. By taking advantage of biophysical and pharmacological diversities of GABA β₂ subunits from the brain and ρ₁ subunits from the retina, structural determinants that confer agonist-induced desensitization were identified. A synthetic chimeric receptor/channel was created from the β₂ and ρ₁ subunits for this investigation. The chimera was constructed from the extracellular N-domain of the β₂ subunit, extending from the amino terminus to the beginning region of the M1 transmembrane segment, and from the C-domain of the ρ₁ subunit extending from the M1 transmembrane segment to the carboxyl terminus. The C-domain region included the M1 to M4 transmembrane regions and the large intracellular loop between the M3 and M4 transmembrane segments. Homo-oligomeric GABA β₂, ρ₁, and β₂/ρ₁ chimeric receptor/channels were individually expressed in Xenopus oocytes, and the desensitization characteristics attributable to each type of subunit were compared. Results from the present study reveal that motifs in the amino-terminal and carboxyl-terminal domains of the β₂ subunit conferred the agonist-induced desensitization; chloroform modulation was linked to specific phases of the GABA-activated current decay. Received: 2 April 1997/Revised: 27 March 1998     

External nickel blocks human Kv1.5 channels stably expressed in CHO cells

We have investigated the actions of Nickel (Ni²⁺) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni²⁺ reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 μm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni²⁺. Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni²⁺, prior to channel activation, was prolonged indicating that Ni²⁺ interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni²⁺ decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni²⁺ preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni²⁺ are distinct from those of other K⁺ channels. Received: 12 October 2000/Revised: 14 May 2001     

Nickel Block of a Family of Neuronal Calcium Channels: Subtype- and Subunit-Dependent Action at Multiple Sites

Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels. To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal calcium channels (α_1A, α_1B, α_1C, and α_1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the activation-curve. Block followed 1:1 kinetics and was most pronounced for α_1C, followed by α_1E > α_1A > α_1B channels. In contrast, the change in activation-gating was most dramatic with α_1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium with calcium reduced both the degree of nickel block and the maximal effect on gating for α_1A channels, but increased the nickel blocking affinity for α_1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α_1A, as coexpression with β_2a or with β₄ resulted in larger current-voltage shifts than those observed in the presence of β_1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively and quantitatively among individual subtypes and subunit combinations. Received: 12 October 1995/Revised: 17 January 1996