The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

We have isolated a novel gene, hKCa4, encoding an intermediate conductance, calcium-activated potassium channel from a human lymph node library. The translated protein comprises 427 amino acids, has six transmembrane segments, S1-S6, and a pore motif between S5 and S6. hKCa4 shares 41-42% similarity at the amino acid level with three small conductance calcium-activated potassium channels cloned from brain. Northern blot analysis of primary human T lymphocytes reveals a 2.2-kilobase transcript that is highly up-regulated in activated compared with resting cells, concomitant with an increase in KCa current. hKCa4 transcript is also detected by Northern blots or by polymerase chain reaction in placenta, prostate, thymus, spleen, colon, and many cell lines of hematopoietic origin. Patch-clamp recordings of hKCa4-transfected HEK 293 cells reveal a large voltage-independent, inwardly rectifying potassium current that is blocked by externally applied tetraethylammonium (K d ‫؍‬ 30 ؎ 7 mM), charybdotoxin (K d ‫؍‬ 10 ؎ 1 nM), and clotrimazole (K d ‫؍‬ 387 ؎ 34 nM), but is resistant to apamin, iberiotoxin, kaliotoxin, scyllatoxin (K d > 1 M), and margatoxin (K d > 100 nM). Single hKCa4 channels have a conductance of 33 ؎ 2 picosiemens in symmetrical potassium solutions. The channel is activated by intracellular calcium (K d ‫؍‬ 270 ؎ 8 nM) with a highly cooperative interaction of approximately three calcium ions per channel. These properties of the cloned channel are very similar to those reported for the native KCa channel in activated human T lymphocytes, indicating that hKCa4 encodes this channel type.Potassium channels play a critical role in modulating calcium signaling of lymphocytes (1). Human T lymphocytes express at least two types of potassium channels (2) (8), and B lymphocytes (9), as well as in other peripheral tissues. However, the molecular identity of this channel type was hitherto unknown. We report the cloning and characterization of an intermediate conductance, CTX-sensitive KCa channel, which we call hKCa4, from a human lymph node cDNA library. We present convergent molecular, biophysical, and pharmacological evidence that hKCa4 encodes the predominant KCa channel in human T cells. EXPERIMENTAL PROCEDURESData Base Search-We performed a BLAST search of a proprietary EST data base (licensed from Incyte Pharmaceuticals, Palo Alto, CA) for unannotated potassium channel sequences using the pore sequence of hKv2.1, a Shab-related K ϩ channel (PASFWWATITMTTVGYGDIYP; Ref. 10). Two overlapping clones of interest were identified, and their sequences were determined (Applied Biosystems PRISM™377 automated sequencer). Both of these clones were from a cDNA library of adherent mononuclear cells, which came from a pool of male and female donors.Library Screening and Computer Analysis-A 32 P-labeled DNA fragment from one of the above clones, corresponding to nucleotides 262-1265 in Fig. 1A, was used as a probe to screen ϳ600,000 recombinant plaques from a human lymph node gt10 cDNA library (CLONTECH). Hybridizations w...

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Expression of functional, recombinant ␣7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor ␣7 subunit. In addition to confirming the previously reported effect on ␣7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional ␣7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface ␣-bungarotoxin binding. Upon application of 1 mM nicotine, currents were undetectable in HEK293 cells expressing only the ␣7 subunit. In contrast, co-expression of ␣7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous ␣7 receptors. Immunoprecipitation studies demonstrate that ␣7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of ␣7 protein on the plasma membrane of cells lacking ric-3, but surface ␣-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of ␣7 receptors in HEK293 cells. Nicotinic acetylcholine receptors (nAChRs)1 are members of the neurotransmitter-gated ion channel superfamily. They are widely expressed in the central and peripheral nervous system (1) where they influence numerous cellular and physiological processes. At least 17 different genes that code for nAChR subunits have been identified (2, 3), and they assemble as pentamers in different combinations to form a diverse set of nAChR subtypes (4, 5). The simplest case is the homopentameric complex such as that formed by the nAChR ␣7 subunit. The ␣7 receptor, for which ␣-bungarotoxin (␣-Bgt) is a specific and high affinity antagonist, is one of the most abundant receptor subtypes in the mammalian brain (6, 7). The high Ca 2ϩ permeability of the ␣7 receptor (8) suggests an involvement in the activation of Ca 2ϩ -dependent events in neurons such as transmitter release, participation in signal transduction, and a variety of modulatory effects (9). In addition, ␣7 receptors have been implicated in a number of diseases such as schizophrenia, Alzheimers, and Parkinsons disease (1, 10 -12).Heterologous expression of the ␣7 subunit in Xenopus oocytes results in homooligomeric, ␣-Bgt-sensitive receptors that activate and inactivate quickly and are highly permeable to Ca 2ϩ (8,13,14), similar to the properties of ␣7 nAChRs in neuronal cells. Although there have been reports of successful functional expression in some mammalian cell lines (15-18), measurable levels of functional receptors have been difficult to achieve in multiple cell types and this phenomenon appears to be host-cell dependent (19). The reasons for poor heterologous surface expression in these cells are not well understood. Strategies to increase the number of functional receptors on the cell...

Abstract--This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at .

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We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca 2؉ -activated K ؉ channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltagegated channel Kv1.3 and the Ca 2؉ -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 Lys 32 , which lies in close proximity to this cluster. These analogs block IKCa1 with ϳ20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 and Kv1.6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-K Ca 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 structurebased strategies to design selective inhibitors for mammalian K ؉ channels. The availability of potent and selective inhibitors of IKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.

Abstract--This summary article presents an overview of the molecular relationships among the voltage-gated sodium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the sodium channel family can be found at .