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R H Joho

Publications and source records attributed to R H Joho.

At least 19 recordsLinked to original sources
PubMed1992

Heterologous expression of the human potassium channel Kv2.1 in clonal mammalian cells by direct cytoplasmic microinjection of cRNA.

The cloned human delayed rectifying K+ channel Kv2.1 (drk1) was expressed in clonal mouse fibroblasts (L-cells) and rat basophilic leukemia cells (RBL-1) by direct cytoplasmic microinjection of complementary RNA (cRNA). Within six hours, cells microinjected with Kv2.1 cRNA expressed a large sustained outward current as determined from whole-cell patch-clamp recordings. Nearly 100% of cells injected with cRNA expressed outward current. Current density was 30-70 pA/pF when measured at a potential of +50 mV. Steady-state activation and inactivation parameters for Kv2.1 were similar when expressed in either L-cells or RBL-1 cells. These results are the first to demonstrate that functional ion channel proteins can be expressed in mammalian clonal cell lines by direct cytoplasmic microinjection of cRNA.

Animals↗
PubMed1992

Differences between the deep pores of K+ channels determined by an interacting pair of nonpolar amino acids.

The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.

Amino Acid Sequence↗
PubMed1992

A single nonpolar residue in the deep pore of related K+ channels acts as a K+:Rb+ conductance switch.

K+ and Rb+ conductances (GK+ and GRb+) were investigated in two delayed rectifier K+ channels (Kv2.1 and Kv3.1) cloned from rat brain and a chimera (CHM) of the two channels formed by replacing the putative pore region of Kv2.1 with that of Kv3.1. CHM displayed ion conduction properties which resembled Kv3.1. In CHM, GK+ was three times greater than that of Kv2.1 and GRb+/GK+ = 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.1 and Kv3.1). A point mutation in CHM L374V, which restored 374 to its Kv2.1 identity, switched the K+/Rb+ conductance profiles so that GK+ was reduced fourfold, GRb+ was increased twofold, and GRb+/GK+ = 2.8. Quantitative restoration of the Kv2.1 K+/Rb+ profiles, however, required simultaneous point mutations at three nonadjacent residues suggesting the possibility of interactions between residues within the pore. The importance of leucine at position 374 was verified when reciprocal changes in K+/Rb+ conductances were produced by the mutation of V374L in Kv2.1 (GK+ was increased threefold, GRb+ was decreased threefold, and GRb+/GK+ = 0.2). We conclude that position 374 is responsible for differences in GK+ and GRb+ between Kv2.1 and Kv3.1 and, given its location near residues critical for block by internal tetraethylammonium, may be part of a cation binding site deep within the pore.

Amino Acid Sequence↗
PubMed1992

Distinct spatial and temporal expression patterns of K+ channel mRNAs from different subfamilies.

Different types of K+ channels play important roles in many aspects of excitability. The isolation of cDNA clones from Drosophila, Aplysia, Xenopus, and mammals points to a large multigene family with several distinct members encoding K+ channels with unique electrophysiological and pharmacological properties. Given the pivotal role K+ channels play in the fine tuning of electrical properties of excitable tissues, we studied the spatial and temporal basis of K+ channel diversity. We report the isolation of two putative K+ channels that define two new subfamilies based upon amino acid sequence similarities with other known K+ channels. Northern blot and in situ hybridization studies revealed differences in the spatial and temporal expression patterns for these two new clones along with mRNAs from other K+ channel subfamilies. Two of the K+ channels studied are predominantly expressed in the brain. One of the "brain-specific" K+ channels is first expressed after about 2 weeks of postnatal cerebellar development and remains at levels about 10-fold higher in the cerebellum than in the rest of the brain.

Amino Acid Sequence↗
PubMed1991

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes.

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K+ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K+ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K+ equilibrium potential (Ek), (2) decreased in slope conductance near Ek, (3) was dependent on [K+]o and (4) was blocked by external Ba2+ and Cs+. These properties were similar to those of the inwardly rectifying K+ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4-5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K+ channels.

Animals↗
PubMed1991

Exchange of conduction pathways between two related K+ channels.

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

Amino Acid Sequence↗
PubMed1991

Electrophysiological characterization of a new member of the RCK family of rat brain K+ channels.

A novel member of the RCK family of rat brain K+ channels, called RCK2, has been sequenced and expressed in Xenopus oocytes. The K+ currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC50 = 3.3 microM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329-3333] but relatively sensitive to 4-aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), alpha-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K+ channel with interesting pharmacological properties.

Amino Acid Sequence↗
PubMed1991

Patterns of internal and external tetraethylammonium block in four homologous K+ channels.

Tetraethylammonium (TEA) is a small ion that is thought to block open K+ channels by binding either to an internal or to an external site. For this reason, it has been used to probe the ion conduction pathway or pore of K+ channel mutants and a K+ channel chimera. The results suggested that the region between transmembrane segments 5 and 6 (S5-S6 linker) was involved in the formation of both the internal and the external TEA binding sites and the K+ conduction pathway. Therefore, we compared internal and external TEA block of the currents expressed in Xenopus oocytes injected with RNAs from four related K+ channel clones, DRK1, RCK1, RCK2, and r-NGK2, which have only subtle structural differences in the S5-S6 linker. r-NGK2 was the most sensitive to external TEA and the least sensitive to internal TEA application. For DRK1 the profile was reversed. RCK1 was blocked equally well from either side, whereas RCK2 was more strongly blocked by internal TEA. The internal block was voltage dependent, whereas the external block was virtually voltage independent. As predicted from block of whole-oocyte currents, internal TEA produced a slow block of DRK1 and RCK2 single-channel currents but had almost no effect on r-NGK2 single-channel currents. Tetrapentylammonium produced a stronger block than TEA at the internal site, and the block was relieved by inward K+ currents, therefore suggesting that the internal TEA binding site is located within the K+ conduction pathway. These results, together with the TEA block of single-channel currents, establish what has until now been inferred by extrapolation from other studies, i.e., that TEA is an open-channel blocker in K+ channel clones. DRK1 mutants with extensive amino- and carboxyl-terminal deletions showed the same blocking profile as the parent DRK1. We conclude that TEA blocks these K+ channels at two sites, which define the inner and outer mouths of the channel pores. Comparison of the primary amino acid sequences in the S5-S6 linker suggests which residues may be responsible for the different patterns of TEA block.

Animals↗
PubMed1990

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker.

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.

Amino Acid Sequence↗
PubMed1990

Isolation of distantly related members in a multigene family using the polymerase chain reaction technique.

We have designed a strategy to isolate and identify genes (cDNAs) coding for distantly-related members within a large multigene family. We have used limited protein sequence information data to delineate conserved regions where members of a supergene family are related. Comparison of the nucleotide sequences of such conserved areas defined consensus sequences that were used for the synthesis of deoxynucleotide primers. Two forward and two reverse primers were synthesized, and four separate pairs of primer combinations were used under low stringency in polymerase chain reactions (PCR) to generate amplified DNA products. The PCR products were directionally cloned into the phage vector M13mp18. Each of four libraries was screened with radiolabeled PCR product generated using a pair of primers different from those used to generate the library. Using this approach on the supergene family of ligand-gated ion channels, we were able to isolate and identify two novel subunits of neurotransmitter-operated ion channels.

Amino Acid Sequence↗
PubMed1990

Toxin and kinetic profile of rat brain type III sodium channels expressed in Xenopus oocytes.

Sodium (Na+) channels are members of a multigene family and are responsible for generation and propagation of the action potential in excitable cells. We have assembled, in a transcription-competent vector, a full-length cDNA clone encoding the rat brain type III Na+ channel. Xenopus oocytes microinjected with in vitro synthesized mRNA expressed functional rat brain Na+ channels from such 'cloned' RNA transcripts. We found that type III Na+ currents in whole cell microelectrode voltage clamp and in cell-attached patch recordings decayed much more slowly than any other reported Na+ current. In addition, we saw typical and additive effects of alpha- and beta-scorpion toxins, suggesting that the Na+ channel alpha-subunit itself contains functional and distinct toxin binding sites.

Amino Acid Sequence↗
PubMed1990

Alteration and restoration of K+ channel function by deletions at the N- and C-termini.

Voltage-dependent ion channels are thought to consist of a highly conserved repeated core of six transmembrane segments, flanked by more variable cytoplasmic domains. Significant functional differences exist among related types of K+ channels. These differences have been attributed to the variable domains, most prominently the N- and C-termini. We have therefore investigated the functional importance of both termini for the delayed rectifier K+ channel from rat brain encoded by the drk1 gene. This channel has an unusually long C-terminus. Deletions in either terminus affected both activation and inactivation, in some cases profoundly. Unexpectedly, more extensive deletions in both termini restored gating. We could therefore define a core region only slightly longer than the six transmembrane segments that is sufficient for the formation of channels with the kinetics of a delayed rectifier.

Animals↗
PubMed1990

Fast and slow gating of sodium channels encoded by a single mRNA.

We investigated the kinetics of rat brain type III Na+ currents expressed in Xenopus oocytes. We found distinct patterns of fast and slow gating. Fast gating was characterized by bursts of longer openings. Traces with slow gating occurred in runs with lifetimes of 5 and 30 s and were separated by periods with lifetimes of 5 and 80 s. Cycling of fast and slow gating was present in excised outside-out patches at 10 degrees C, suggesting that metabolic factors are not essential for both forms of gating. It is unlikely that more than one population of channels was expressed, as patches with purely fast or purely slow gating were not observed. We suggest that structural mechanisms for fast and slow gating are encoded in the primary amino acid sequence of the channel protein.

Animals↗
PubMed1990

Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor.

Thyrotropin-releasing hormone (TRH) is an important extracellular regulatory molecule that functions as a releasing factor in the anterior pituitary gland and as a neurotransmitter/neuromodulator in the central and peripheral nervous systems. Binding sites for TRH are present in these tissues, but the TRH receptor (TRH-R) has not been purified from any source. Using Xenopus laevis oocytes in an expression cloning strategy, we have isolated a cDNA clone that encodes the mouse pituitary TRH-R. This conclusion is based on the following evidence. Injection of sense RNA transcribed in vitro from this cDNA into Xenopus oocytes leads to expression of cell-surface receptors that bind TRH and the competitive antagonist chlordiazepoxide with appropriate affinities and that elicit electrophysiological responses to TRH with the appropriate concentration dependency. Antisense RNA inhibits the TRH response in Xenopus oocytes injected with RNA isolated from normal rat anterior pituitary glands. Finally, transfection of COS-1 cells with this cDNA leads to expression of receptors that bind TRH and chlordiazepoxide with appropriate affinities and that transduce TRH stimulation of inositol phosphate formation. The 3.8-kilobase mouse TRH-R cDNA encodes a protein of 393 amino acids that shows similarities to other guanine nucleotide-binding regulatory protein-coupled receptors.

Amino Acid Sequence↗
PubMed1989

A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning.

Voltage-activated potassium channels play an important part in the control of excitability in nerve and muscle. Different K+ channels are involved in establishing the resting potential, determining the duration of action potentials, modulation of transmitter release, and in rhythmic firing patterns and delayed excitation. Using in vitro transcripts made from a directional complementary DNA library we have isolated, by expression cloning in Xenopus oocytes, a novel K+-channel gene (drk1). Functionally, drk1 encodes channels that are K+ selective and belong to the delayed rectifier class of channels, rather than the A-type class encoded by the Shaker gene of Drosophila. The channels show sigmoidal voltage-dependent activation and do not inactivate within 500 ms. Structurally, drk1 encodes an amino-acid sequence which is more closely related to the Drosophila Shab gene than to the Shaker gene.

Amino Acid Sequence↗
PubMed1987

T-cell antigen receptors with identical variable regions but different diversity and joining region gene segments have distinct specificities but cross-reactive idiotypes.

The T-cell antigen receptor alpha-chain genes of an alloreactive, H-2Db-specific cytotoxic T-cell clone (3F9) are described. This study and our work on the 3F9 beta-chain genes reveal that the variable region gene segments for the alpha and beta chains expressed in 3F9 are identical to the ones used by a chicken erythrocyte-specific, I-Ab-restricted helper T-cell clone (LB2). These two clones differ, however, in the diversity and joining portions of the alpha and beta chains of their T-cell receptor molecules. The analysis of 3F9 and LB2 with monoclonal antibodies specific for the 3F9 T-cell receptor shows that these two T-cell clones share the same idiotype; however, 3F9 and LB2 do not exhibit any antigen and/or major histocompatibility complex cross-reactivity. This suggests that the diversity and joining regions of the T-cell receptor may play a key role in antigen and/or major histocompatibility complex recognition.

Animals↗