K. G. Chandy

A voltage-gated potassium channel in human T lymphocytes.

Human peripheral T lymphocytes were studied at 20‐24 degrees C using the gigaohm seal recording technique in whole‐cell or outside‐out patch conformations. The predominant ion channel present under the conditions employed was a voltage‐gated K+ channel closely resembling delayed rectifier K+ channels of nerve and muscle. The maximum K+ conductance in ninety T lymphocytes ranged from 0.7 to 8.9 nS, with a mean of 4.2 nS. The estimated number of K+ channels per cell is 400, corresponding to a density of about three channels/micron2 apparent membrane area. The activation of K+ currents could be fitted by Hodgkin‐Huxley type n4 kinetics. The K+ conductance in Ringer solution was half‐maximal at ‐40 mV. The time constant of K+ current inactivation was practically independent of voltage except near the threshold for activating the K+ conductance. Recovery from inactivation was slow and followed complex kinetics. Steady‐state inactivation was half‐maximal at ‐70 mV, and was complete at positive potentials. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+(1.0) greater than Rb+(0.77) greater than NH4+(0.10) greater than Cs+ (0.02) greater than Na+(less than 0.01). Currents through K+ channels display deviations from the independence principle. The limiting outward current increases when external K+ is increased, and Rb+ carries less inward current than expected from its relative permeability. Tail current kinetics were slowed about 2‐fold by raising the external K+ concentration from 4.5 to 160 mM, and were 5 times slower in Rb+ Ringer solution than in K+ Ringer solution. Single K+ channel currents had two amplitudes corresponding to about 9 and 16 pS in Ringer solution. Replacing Ringer solution with isotonic K+ Ringer solution increased the unitary conductance and resulted in inward rectification of the unitary current‐voltage relation. Comparable effects of external K+ were seen in the whole‐cell conductance and instantaneous current‐voltage relation. Several changes in the K+ conductance occurred during the first few minutes after achievement of the whole‐cell conformation. Most are explainable by dissipation of a 10‐20 mV junction potential between pipette solution and the cytoplasm, and by the use of a holding potential more negative than the resting potential. However, inactivation of K+ currents became faster and more complete, changes not accounted for by these mechanisms. K+ efflux through open K+ channels in intact lymphocytes, calculated from measured properties of K+ channels, can account for efflux values reported in resting lymphocytes, and for the increase in K+ efflux upon mitogenic stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

The Signature Sequence of Voltage-gated Potassium Channels Projects into the External Vestibule

A highly conserved motif, GYGD, contributes to the formation of the ion selectivity filter in voltage-gated K+ channels and is thought to interact with the scorpion toxin residue, Lys27. By probing the pore of the Kv1.3 channel with synthetic kaliotoxin-Lys27 mutants, each containing a non-natural lysine analog of a different length, and using mutant cycle analysis, we determined the spatial locations of Tyr400 and Asp402 in the GYGD motif, relative to His404 located at the base of the outer vestibule. Our data indicate that the terminal amines of the shorter Lys27 analogs lie close to His404 and to Asp402, while Lys27 itself interacts with Tyr400. Based on these data, we developed a molecular model of this region of the channel. The junction between the outer vestibule and the pore is defined by a ring (∼8-9-Å diameter) formed from alternating Asp402 and His404 residues. Tyr400 lies 4-6 Å deeper into the pore, and its interaction with kaliotoxin-Lys27 is in competition with K+ ions. Studies with dimeric Kv1.3 constructs suggest that two Tyr400 residues in the tetramer are sufficient to bind K+ ions. Thus, at least part of the K+ channel signature sequence extends into a shallow trough at the center of a wide external vestibule.

Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca2+-activated over Voltage-gated K+ Channels

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca2+-activated K+channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3and the Ca2+-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys32, which lies in close proximity to this cluster. These analogs block IKCa1with ∼20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 andKv1.6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-KCa channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K+channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K+channels. The availability of potent and selective inhibitors ofIKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.

Purification, Visualization, and Biophysical Characterization of Kv1.3 Tetramers

The voltage-gated K+ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 104 to 5 × 105 functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a ∼2-kDa core-sugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of ∼64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the ∼64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of ∼65 × 65 Å. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K+-selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.

The P-region and S6 of Kv3.1 contribute to the formation of the ion conduction pathway.

The loop between transmembrane regions S5 and S6 (P-region) of voltage-gated K+ channels has been proposed to form the ion-conducting pore, and the internal part of this segment is reported to be responsible for ion permeation and internal tetraethylammonium (TEA) binding. The two T-cell K+ channels, Kv3.1 and Kv1.3, with widely divergent pore properties, differ by a single residue in this internal P-region, leucine 401 in Kv3.1 corresponding to valine 398 in Kv1.3. The L401V mutation in Kv3.1 was created with the anticipation that the mutant channel would exhibit Kv1.3-like deep-pore properties. Surprisingly, this mutation did not alter single channel conductance and only moderately enhanced internal TEA sensitivity, indicating that residues outside the P-region influence these properties. Our search for additional residues was guided by the model of Durell and Guy, which predicted that the C-terminal end of S6 formed part of the K+ conduction pathway. In this segment, the two channels diverge at only one position, Kv3.1 containing M430 in place of leucine in Kv1.3. The M430L mutant of Kv3.1 exhibited permeant ion- and voltage-dependent flickery outward single channel currents, with no obvious changes in other pore properties. Modification of one or more ion-binding sites located in the electric field and possibly within the channel pore could give rise to this type of channel flicker.

Translation Initiation of a Cardiac Voltage-gated Potassium Channel by Internal Ribosome Entry

The mammalian Kv1.4 voltage-gated potassium channel mRNA contains an unusually long (1.2 kilobases) 5′-untranslated region (UTR) and includes 18 AUG codons upstream of the authentic site of translation initiation. Computer-predicted secondary structures of this region reveal complex stem-loop structures that would serve as barriers to 5′ → 3′ ribosomal scanning. These features suggested that translation initiation in Kv1.4 might occur by the mechanism of internal ribosome entry, a mode of initiation employed by a variety of RNA viruses but only a limited number of vertebrate genes. To test this possibility we introduced the 5′-UTR of mouse Kv1.4 mRNA into the intercistronic region of a bicistronic vector containing two tandem reporter genes, chloramphenicol acetyltransferase and luciferase. The control construct translated only the upstream chloramphenicol cistron in transiently transfected mammalian cells. In contrast, the construct containing themKv1.4 UTR efficiently translated the luciferase cistron as well, demonstrating the presence of an internal ribosome entry segment. Progressive 5′ → 3′ deletions localized the activity to a 3′-proximal 200-nucleotide fragment. Suppression of cap-dependent translation by extracts from poliovirus-infected HeLa cells in anin vitro translation assay eliminated translation of the upstream cistron while allowing translation of the downstream cistron. Our results indicate that the 5′-untranslated region ofmKv1.4 contains a functional internal ribosome entry segment that may contribute to unusual and physiologically important modes of translation regulation for this and other potassium channel genes.

Autologous mixed lymphocyte reaction in man: XV. Cellular and molecular basis of deficient autologous mixed lymphocyte response in insulin-dependent diabetes mellitus.

The autologous mixed lymphocyte response (AMLR) and the allogeneic mixed lymphocyte response were deficient in a subset of patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM). Using a single set of HLA-identical twins, the cellular and molecular basis of deficient AMLR was investigated and appears to be due to a defect in both responder T cells and stimulator non-T cells. Interleukin-2 production was diminished in the patient but not in the healthy twin. Thein vitro addition of purified interleukin-2 enhanced the depressed AMLR in the diseased twin. This suggests that the deficient AMLR in IDDM may be in part due to a deficiency in the production of interleukin-2.

Ion channels in lymphocytes

The advent of the gigaohm-seal recording technique has enabled the study of the electrical properties of small cells, such as individual lymphocytes. Recent studies using this technique in combination with standard immunological and biochemical techniques indicate that cells of the immune system may utilize ion channels, similar in properties to those described in nerve and muscle, in the process of activation. For example, potassium channels may be required for T-lymphocyte mitogenesis and calcium channels for antibody production. This article summarizes these recent reports.

Chapter 11 Potassium Channels in Development, Activation, and Disease in T Lymphocytes

Publisher Summary In the nervous system, voltage-gated Na + and K + channels effect the upstroke and repolarizing phases, respectively, of the action potential. Cells of the immune system do not generate action potentials. Depolarization that induces Ca 2+ signals in electrically excitable nerve and muscle cells inhibits Ca 2+ signaling and the subsequent cascade of cell-activation events in lymphocytes. Several distinct types of voltage-gated and second-messenger-operated K + and C1 − channels exist in T and B lymphocytes, including channel types also expressed in the nervous system, as well as novel channels not described in other cell types. In electrically inexcitable cells, ion channels mediate cellular functions, involving intracellular biochemical signaling. The presence of K + channels is apparently required for several basic functions in T lymphocytes. There is diversity of ion channels within cells of the hematopoietic lineage. In T lymphocytes, at least eight distinct types of K + , C1 − , and Ca 2+ channels have been characterized. Investigators have also recorded from bone marrow cells, thymocytes, B lymphocytes, monocytes, macrophages, neutrophils, red blood cells, eosinophils, and platelets, as well as numerous related cell lines, including a human T-leukemia line, a mouse T-lymphoma line (S49), a variety of antibody-secreting hybridomas, and a mast-cell line derived from a rat basophilic leukemia (RBL). These studies have revealed transient or T-type voltage-gated Ca 2+ channels (hybridomas), inward-rectifying K + channels (macrophages, RBL cells), large-conductance Ca 2+ -activated K + channels (macrophages), Ca 2+ -activated C1 − current (mast cells), and G-protein-activated K + channels (RBL cells). In this chapter, K + channels in T lymphocytes also containing a summary of the properties of known voltage- and Ca 2+ -activated K + channels in lymphocytes and their molecular characteristics, functions, and expression during normal development, mitogen activation, and in autoimmune disorders are discussed. In addition, the chapter describes the coordinated activity of Ca 2+ and K + channels that underlies the mitogen-stimulated Ca 2+ signaling.