Natural Substitutions at Highly Conserved T1-Domain Residues Perturb Processing and Functional Expression of Squid Kv1 Channels

Taylor I. Liu,1 Zora N. Lebaric,2 Joshua J. C. Rosenthal,2 and William F. Gilly1,2

 1Neurosciences Program and  2Department of Biological Sciences, Hopkins Marine Station, Stanford University, Pacific Grove, California 93950


    ABSTRACT
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Liu, Taylor I., Zora N. Lebaric, Joshua J. C. Rosenthal, and William F. Gilly. Natural Substitutions at Highly Conserved T1-Domain Residues Perturb Processing and Functional Expression of Squid Kv1 Channels. J. Neurophysiol. 85: 61-71, 2001. Shaker-type K-channel alpha -subunits (SqKv1A, B, D) expressed in neurons of the squid stellate ganglion differ in the length of their N-termini and in the species of amino acid present at several points in the T1 domain, an intracellular region involved in the tetramerization process during channel assembly. Heterologous expression of wild-type SqKv1A, B, and D in Xenopus oocytes reveals large differences in the level of both functional channels (assayed by whole-oocyte voltage clamp) and total channel protein (assayed by immunoblotting). Functional expression is poorest with SqKv1A and by far the best with SqKv1D. Biophysical properties of the three SqKv1 channels are essentially identical (assayed by cell-attached patch clamp). Site-directed mutagenesis was used to determine whether the observed differences in expression level are impacted by two residues in the T1 domain at which SqKv1A and B (but not D) differ from the consensus sequences found in many other taxa. In SqKv1A, glycine is substituted for arginine in an otherwise universally conserved sequence (FFDR in the T1B subdomain). In SqKv1B, glycine replaces serine in a sequence that is conserved within the Kv1 subfamily (SGLR in the T1A subdomain). Restoration of the consensus amino acid at these positions largely accounts for the observed differences in expression level. Analysis of the glycosylation state of aberrant versus restored alpha -subunits suggests that the anomalous amino acids in SqKv1A and B exert their influence during early steps in channel processing and assembly which take place in the endoplasmic reticulum (ER).


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INTRODUCTION
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A defined segment of the cytoplasmic amino- (N-) terminus of voltage-gated K (Kv) channels, known as T1 or NAB, contains contact points at which alpha -subunits oligomerize into tetrameric channels (Li et al. 1992; Shen et al. 1993). Two important subdomains, T1A and T1B, have been identified within T1. Conserved "distinct" sequences in T1B were originally noted by Wei et al. (1990) and distinguish the Kv1-4 subfamilies (starred brackets in Fig. 1B). Members of different subfamilies do not form heteromultimeric channels (Covarrubias et al. 1991; Xu et al. 1995), and this incompatibility is contributed to by both subdomains (Shen and Pfaffinger 1995). Consequently, the T1 domain confers intra-subfamily specificity to the tetramerization of Kv alpha -subunits and thereby directly influences the complement of functional Kv channels expressed in an individual cell.



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Fig. 1. Comparison of features of the N-terminal regions of SqKv1A-D. A: alignment of amino acids upstream of the T1 domain is illustrated with dashes representing identities. The general locations of 7 other sites where differences occur in T1 and C-terminal domains are indicated by diamonds. There are no amino acid differences within the core region. B: alignment of partial T1 domain sequences of SqKv1 isoforms with selected other Kv1, Kv2, Kv3, and Kv4 alpha -subunits. The A and B subdomains are indicated by dark brackets (T1A, T1B), and the Kv1-specific sequences of Wei et al. (1990) are marked by asterisks and light brackets. Dashes in SqKv1 sequences represent identities to SqKv1A; empty spaces are gaps introduced by the alignment. Bold letters indicate the two highly conserved stretches (SGLR and FFDR) discussed in this paper in which marked deviations (arrows) from the consensus occurs in SqKv1A (G87) and SqKv1B (S47 position of SqkV1A).

We have previously described three cDNAs that encode Kv1 alpha -subunits expressed in the squid giant axon system (SqKv1A; Rosenthal et al. 1996) and in other, nongiant neurons of the stellate ganglion (SqKv1B and D; Rosenthal et al. 1997). All three isoforms share a common core region (S1-S6) but differ in the length and composition of the N-terminal domains (Fig. 1A) with five differences in the T1 region (Fig. 1B; numbering for SqKv1A).

Two of these five differences occur in short stretches of amino acids that are extremely conserved in other Kv members. The T1B sequence FFDR is present in every entry in Fig. 1B (bold) with the exception of SqKv1A, in which the consensus arginine is replaced by glycine (G87). A second highly conserved sequence is SGLR in T1A (Fig. 1B), which differentiates Kv1 members from those of the other subfamilies. SqKv1B lacks the consensus serine (S47 in SqKv1A), which again is replaced by glycine. This residue occupies a structurally important position in the "tetramerization interface" of the T1 domain of an Aplysia Kv1 alpha -subunit (AKv1.1a or AK01A) and may play a critical role in preventing tetramerization of Kv1 and Kv3 alpha -subunits (Bixby et al. 1999; Kreusch et al. 1998).

These aberrant amino acids in SqKv1A (G87) and SqKv1B (G36) may be expected to influence tetramerization and possibly other steps in channel assembly. In this paper, we explore the impact of these residues on expression of SqKv1 channels in Xenopus oocytes and make use of SqKv1D, which maintains the consensus amino acid at both positions, as a standard for comparison. In each case, the anomalous amino acid greatly reduces the density of functional K channels assayed by two-electrode voltage clamp as well as the level of mature, fully glycoslyated alpha -subunit protein determined by immunoblotting. Biochemical analysis of normal and defective alpha -subunits suggests that factors leading to poor expression act early in the biosynthetic pathway, probably in the endoplasmic reticulum (ER). Although glycosylation is not necessary to form functional SqKv1 channels, our results indicate that it may increase the efficiency of surface expression at early times following cRNA injection.


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Construction of expression plasmids and site-directed mutagenesis

SQKV1A, B, AND D. Full-length cDNAs were isolated from a squid stellate ganglion library as previously described (Rosenthal et al. 1996, 1997). Polymerase chain reaction (PCR) was used to amplify the entire coding region of each cDNA with primers containing Bgl II sites flanking the start and stop codons. SqKv1A-D have the identical C-terminal, and a common antisense primer was employed (SKC 16 of Rosenthal et al. 1997 and Table 1). The sense primers were designed to generate a consensus translation initiation sequence (Kozak 1989). For SqKv1A (SKC37, Table 1), only the nucleotide at the -3 position was changed (C to A), and no amino acid (aa) codons were thereby altered. In the cases of SqKv1B and D, however, an additional change at the +4 position (T to G) resulted in alteration of aa 2 from Ser to Ala and from Tyr to Asp, respectively.


                              
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Table 1. Oligonucleotides used for PCR amplification and cloning procedures

The above products were sub-cloned and ligated into the Bgl II site of pBSTA, an expression vector that contains 5' and 3' Xenopus beta -globin untranslated regions. In most cases, the High Fidelity PCR System (Boehringer Mannheim, Indianapolis, IN) was used. If PCR was carried out with Taq polymerase, a large Nde I-Spe I fragment (1045 nt) of the cloned amplification product was replaced with the corresponding fragment from the original cDNA, and the remaining portions were confirmed by manual sequencing (Sequenase 2.0 Kit; US Biochem). Sequences through the ligation sites in all constructs described were also confirmed in this manner.

SQKV1A Delta V5. Three nucleotides (GTT) encoding Val at position 5 of SqKv1A were omitted from the 5' (sense) primer SCK37; this primer was thus SKC 15 used in a previous study (Rosenthal et al. 1996). Amplification thus introduced the Delta V5 mutation into the full-length product which was then ligated into pBSTA as described above.

SQKV1A G87R, SQKV1B R76G, AND SQKV1D R53G. A BstE II-Kas I fragment (109 nt) was removed from SqKv1A-pBSTA and replaced with the corresponding portion of SqKv1B. This changed the codon for aa 87 of SqKv1A from GGG (Gly) to AGG (Arg); the fragments were otherwise identical. To replace Arg with Gly at the equivalent position in SqKv1B-pBSTA (aa 76) and SqKv1D-pBSTA (aa 53), the same fragment was replaced with that from SqKv1A.

SQKV1A Delta 1-34 G87R. This mutant was created by replacing most of the coding region of SqKv1D-pBSTA [from aa 4 (RsrII site) to the stop codon (Bgl II)] with the corresponding fragment of SqKv1A G87R-pBSTA. The resulting construct (called Delta 1-34 G87R for simplicity) lacks residues 1-34 and begins with the initiating Met of SqKv1D. Cysteine at position 36 (i.e., aa 2 of the mutant) was changed by this approach to Asp in order to maintain a Kozak consensus sequence as described above.

SQKV1B G36S AND SQKV1D S13G. A Rsr II-Kas I fragment (195 nt) was removed from SqKv1B-pBSTA and replaced with the corresponding fragment of SqKv1D. This changed the codon for aa 36 of SqKv1B from GGC (Gly) to AGC (Ser); the fragments were otherwise identical. SqKv1D S13G was created by removing the same fragment in SqKv1D-pBSTA and replacing it with that from SqKv1B.

SQKV1A G87R HA-TAG. An in-frame, HA-epitope tag (YPYDVPDYASL) was incorporated into the C-terminal of SqKv1A G87R as follows. Hind III and BamH I sites were introduced into SqKv1A G87R before the start and stop codons, respectively, by PCR (sense primer SKC38 and antisense primer SKC36, Table 1) using SqKv1A G87R-pBSTA as a template. This amplification product was ligated into the Hind III and BamH I sites of the vector pOX (gift of T. Jegla, Stanford University), which contains the same Xenopus-untranslated regions as pBSTA. A synthetic, double-stranded oligonucleotide encoding the HA epitope with a BamH I site at the 5' end and a Xho I site at the 3' end was ligated into the same sites in SqKv1A G87R-pOX.

SQKV1A-D GLYCOSYLATION MUTANTS. All glycosylation mutations (Asp to Gln) were made using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Mutations were confirmed by sequencing.

Xenopus oocyte preparation and cRNA injection

Stage IV oocytes were isolated from adult Xenopus laevis and treated at room temperature with 1.5 mg/ml collagenase Type IA (Sigma, St. Louis, MO) in Ca-free OR-2 [(in mM) 82 NaCl, 2.5 KCl, 1 MgCl2, 5 HEPES; pH 7] until single oocytes were released (~2 h). Oocytes were then washed (room temperature) three times in Ca-free OR-2 and three times with ND96 [(in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES; pH 7.2] before incubation at 18°C in supplemented ND96 containing 2.5 mM Na-pyruvate and 50 µg/ml gentamycin. cRNA injection was carried out 12-24 h later.

Full-length cRNAs were transcribed from SqKv1 expression plasmids linearized with Not I using either T7 (for pBSTA) or T3 (for pOX) RNA polymerase (Message Machine, Ambion, Austin, TX). Products were quantified by absorbance at 260 nm prior to injection, and comparative concentrations were also checked by ethidium bromide staining of samples in 1% TAE gels. Oocytes were injected with 8 ng of cRNA (50 nl vol) through a glass micropipette using a manual microsyringe (Drummond Scientific, Broomall, PA) and were then cultured in supplemented ND96 as described above until recordings were carried out. Medium changes were made daily.

Tissue preparation and immunoblotting

Oocytes were harvested for analysis 2 days after cRNA injection. Five to six oocytes were homogenized using a Dounce homogenizer in ice-cold lysis buffer [(in mM) 5 Tris-HCl, 1 EDTA, 1 EGTA; pH 8] containing freshly added protease inhibitors {1 µg/ml each of aprotinin, leupeptin, and pepstatin A plus 50 mg/ml AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] (all from Sigma)}. Yolk and cellular debris were cleared from the homogenate by centrifugation at 3000 g for 10-15 min at 4°C. A crude membrane fraction was then pelleted by another centrifugation at 100,000 g for 30 min at 4°C. This pellet was resuspended in 12 µl SDS sample buffer (Sambrook et al. 1989) plus 0.15 M beta -mercaptoethanol. Samples were heated to 65°C for 5 min, separated using SDS-PAGE on 7.5% gels, and electrophoretically transferred to nitrocellulose membranes which were then soaked for 2 h at room temperature in 10% nonfat dry milk in PBS [(in mM) 80 Na2HPO4, 20 NaH2PO4, 100 NaCl; pH 7.4]. Affinity-purified polyclonal antibodies to SqKv1A (Rosenthal et al. 1996) were used at 1:2000 dilution in PBS with overnight incubation at 4°C. Goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Sigma, St. Louis, MO) were used at 1:5000 dilution with a 45-60 min incubation time at room temperature. An enhanced chemiluminescence detection system (Super Signal Substrate, Pierce, Rockford, IL) was used to visualize the signals.

Immunoprecipitation and endoglycosidase digestion

These experiments employed the SqKv1A G87R HA-tag construct. After oocytes were injected with cRNA, they were maintained in supplemented ND96 containing 500 µCi/ml [35S]-methionine (Amersham) for 2 days at 18°C. Ten oocytes were collected and processed as described by Ivanina et al. (1994) with minor modifications. Primary antibody (monoclonal anti-HA, Berkeley Antibody Co., Berkeley, CA) was used at a dilution of 1:150. Secondary antibodies coupled to magnetic beads (BioMag goat anti-mouse IgG, PerSpective Diagnostics, Cambridge, MA) were used at a final dilution of 1:200. Immunoprecipitated proteins coupled to the magnetic beads were suspended in 15 µl SDS sample buffer containing 0.15 M beta -mercaptoethanol, heated to 65°C for 5 min, and loaded onto 7.5% SDS-PAGE. Gels were fixed in 45% methanol-10% glacial acetic acid (10-15 min), treated for 30 min with autoradiography enhancer (Enhance, NEN, Boston, MA), washed in distilled H2O (10-15 min), dried, and exposed to autoradiography film (X-OMAT AR, Kodak) overnight at -70°C.

For endoglycosidase digestion, magnetic beads were resuspended in 10 µl wash buffer (Ivanina et al. 1994) after the final wash. Proteins attached to beads were denatured by heating to 100°C for 10 min. Digestion with endoglycosidase H (New England Biolabs, Beverly, MA) was then carried out according to manufacturer's instructions at 37°C for 1.5 h. SDS sample buffer with 0.15 M beta -mercaptoethanol was added before continuing with gel electrophoresis as described above.

Two-microelectrode whole-oocyte voltage clamp

Recordings were made in ND96 at room temperature (22-25°C) using a conventional amplifier (OC-725B, Warner Instruments, Hamden, CT) and procedures. Microelectrodes were filled with 3 M KCl and had resistances of about 1 MOmega . Analog K current (IK) signals were filtered at 5 kHz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Data acquisition, pulse generation, and on-line signal processing (P/-4 treatment to remove linear leakage and capacity currents) were carried out using a system designed by Dr. D. R. Matteson, University of Maryland School of Medicine.

Maximum K slope conductance (gK) was calculated for individual oocytes from the peak IK-voltage (V) relation between 0 and +60 mV using a linear regression fit. The gK-V relation for all channels studied here is saturated over this range (Rosenthal et al. 1997 and unpublished data). In some cases when IK was extremely large, IK at +10 mV was taken as an index of gK.

Cell-attached macropatch recordings

All recordings were conducted 2-4 days after cRNA injection as previously described (Rosenthal et al. 1997). The vitelline membrane was manually removed after soaking an oocyte for several minutes in a hypertonic solution containing (in mM) 200 K-aspartate, 20 KCl, 10 EGTA, 1 MgCl2, 10 HEPES; pH 7.4. The oocyte was then immediately transferred to the recording chamber filled with a bath solution [(in mM) 140 KCl, 2 MgCl2, 11 EGTA, 1 CaCl2, 10 HEPES; pH 7.2], in which all recordings were carried out at room temperature. Pipettes (1-3 MOmega ) were coated with Sylgard (Dow Corning, Midland, MI) and filled with external solution [(in mM) 127 NaCl, 15 KCl, 6 MgCl2, 1 CaCl2, 5 HEPES; pH 7.1]. After attaining a suitable high-resistance seal, the stability of IK was verified over a 10 min period before acquiring data for analysis. Signals were filtered (15 kHz), sampled, and processed (P/-4) as described above.


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SqKv1 isoforms are expressed at different levels in Xenopus oocytes

Oocytes were injected on day 0 with an identical amount (8 ng) of cRNA encoding either SqKv1A, B, or D alpha -subunits, and gK was determined from five oocytes of each type over the next 5 days. Although the time course of gK increase is roughly similar in each case, the maximum level of gK attained over days 3-4 differs tremendously (Fig. 2). SqKv1D channels are expressed at a level about four times greater than that of SqKv1B and roughly 80 times that of SqKv1A.



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Fig. 2. Time course of K conductance (gK) development over time in oocytes injected with 8 ng of cRNA encoding either SqKv1A, B, or D. Conductance was calculated from the slope of the IK-voltage relation over the range of 0 to +60 mV. Each point represents the mean ± SE from 5 oocytes. At the time of peak expression (days 3-4), gK for SqKv1D (triangles) is ~4 times that for SqKv1B (squares) and nearly 100 times that for SqKv1A (circles).

Identification of N-terminal elements in SqKv1A that decrease functional expression

The primary structure of SqKv1A, the most poorly expressed isoform, shows two obvious differences from that of SqKv1D which could be relevant to the low level of functional expression observed. First, the N-terminal of SqKv1A is substantially longer and unusually hydrophobic (8 of the first 11 amino acids). Second, of the four amino acids that differ in the T1 domain (Fig. 1B), G87 stands out as the only major anomaly in SqKv1A. This isoform contains the overwhelming consensus residue displayed by other Kv1 members at two of the other sites (V43 and N45), and the third site (K132) is not highly conserved. In an effort to increase the expression level shown by SqKv1A to that of SqKv1D, we therefore individually altered these two features.

ALTERATION OF G87 TO THE ARG CONSENSUS. Changing Gly 87 in SqKv1A to Arg dramatically affects the level of functional expression in oocytes without altering voltage-dependent properties of gK or single-channel conductance. Two-microelectrode recordings from oocytes injected with equal amounts of wild-type SqKv1A or G87R cRNA are displayed in Fig. 3, A and B. Activation kinetics and the gK-V relation (data not illustrated) are very similar in both cases. However, IK at +10 mV, a good indicator of maximum gK, is greatly increased by the G87R mutation (Fig. 3D).



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Fig. 3. Effects of altering the anomalous G87 residue in T1 and deleting the extreme N-terminus on functional expression of SqKv1A. Two-electrode recordings were made 3 days after cRNA injection. A: representative IK records for wild-type SqKv1A are illustrated at the indicated voltages. B and C: families of IK records (same voltages as in A) for SqKv1A with the G87R mutation (B) and with deletion of the N-terminus (Delta 1-34) in the G87R background (C). No large differences in functional properties of these constructs are evident. D: functional expression is compared for the above constructs and SqKv1D. Mean IK (±SE) at +10 mV was computed for 5 oocytes of each type (see also METHODS). Restoring the consensus Arg87 (A G87R) produces nearly an ~20-fold increase in functional expression over that seen with wild-type SqKv1A (A). Deletion of the N-terminal of the former construct (A Delta 1-34 G87R) restores expression to a level comparable to that for SqKv1D.

Single-channel recordings from cell-attached patches from oocytes expressing SqKv1A G87R channels (data not illustrated) showed that the unitary conductance (~14 pS) and peak open probability at positive voltages (~0.9) are comparable to those observed for with Gly at position 87 (Rosenthal et al. 1996). In the latter case, however, the actual "wild-type" alpha -subunit also had the Delta V5 mutation, which itself increases expression level. Although the very low level of IK seen with true wild-type SqKv1A precludes single-channel recordings, unitary conductance, and peak open probability for SqKv1A G87R channels that also have the Delta V5 mutation are comparable to those indicated above (unpublished data). Thus, the increased level of gK due to the G87R mutation reflects an increase in the surface density of functional SqKv1A channels.

DELETION OF THE N-TERMINAL. Although the G87R mutation increases functional expression, the maximum level of gK attained is still substantially less than that seen with expression of SqKv1D (Fig. 3D). To test the idea that the longer, hydrophobic N-terminus of SqKv1A could have a deleterious effect on functional expression, the first 34 amino acids of SqKv1A G87R were deleted in a way that made the N-terminus identical to that of SqKv1D (Delta 1-34 G87R; see METHODS). This manipulation increased functional expression to the level observed for SqKv1D (Fig. 3D), again with no significant effects on voltage-dependent properties (Fig. 3C).

DELETION OF VAL 5. Although we have not carried out an extensive analysis of the N-terminal elements of SqKv1A that impair functional expression, we have identified a single amino acid that is important. Deletion of Val at position 5 in the wild-type SqKv1A (SqKv1A Delta V5) leads to an increase in functional expression of about fourfold (Fig. 4D) with no apparent change in voltage-dependent properties (Fig. 4, A and B). Data in Fig. 4D indicate that the Delta V5 mutation in the G87R background (SqKv1A Delta V5 G87R) increases the level of functional expression to the same degree as that observed with the complete deletion of the N-terminal (Fig. 3D). Replacement of Val with other amino acids or additional deletions were not explored.



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Fig. 4. Deletion of the N-terminal of SqKv1A is mimicked by deletion of Val 5. Data were obtained as described in conjunction with Fig. 3 and are presented in an analogous format. A: IK records obtained with wild-type SqKv1A are replotted from Fig. 3 for comparative purposes. B: IK records for SqKv1A with Val 5 deleted (Delta V5) are larger in amplitude than those of wild-type channels but are otherwise similar. C: IK for the Val 5 deletion in the G87R background is restored to a very high level. D: mean IK (±SE) at +10 mV seen with wild-type SqKv1A (A) is increased ~4-fold by deleting Val 5 (A Delta V5) and increased nearly 20-fold more when this mutation is combined with restoring the consensus Arg 87 (A Delta V5 G87R).

Voltage-dependent macroscopic properties of the Delta V5 G87R channels appear to be normal (Fig. 4C), and as discussed above, unitary conductance and peak open probability is not changed by the G87R mutation. Thus, we interpret the 80-fold increase in IK (+10 mV) between wild-type SqKv1A and SqKv1A Delta V5 G87R as being due to an increase in functional channel density in the surface membrane.

Voltage-dependent properties of SqKv1A Delta V5 G87R and SqKv1D

The combined Delta V5 and G87R mutations in SqKv1A restores functional expression to the high level observed for SqKv1D, and this permits reliable patch-clamp analysis of macroscopic voltage-dependent properties of IK through these SqKv1 channels. This is not possible with constructs that express at much lower levels. Although the identical core structure of these alpha -subunits suggests the corresponding channels should have very similar properties, this is an important point to confirm.

Basic voltage-dependent properties of SqKv1A Delta V5 G87R channels derived from cell-attached patch recordings are compared with those of SqKv1D in Fig. 5. IK records for both types of channels illustrate the similar natures of activation (Fig. 5A), deactivation (Fig. 5B), and inactivation (Fig. 5C). A population analysis indicates that gK-V relations for the two channels are not significantly different (Fig. 5D), and the same is true for activation kinetics (Fig. 5E), deactivation kinetics (Fig. 5F), and inactivation kinetics (Fig. 5G).



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Fig. 5. Comparison of functional properties of SqKv1A Delta V5 G87R [(i) in panels A-C] and SqKv1D [(ii) in panels A-C] studied in cell-attached patches. A: representative IK records (-20, 0, 20, 40 mV) for each channel type reveal no substantial differences in activation kinetics. B: superimposed IK tail traces (-140, -110, and -80 mV) show no differences in deactivation kinetics between the two channel types. Activating pulses were to +40 mV for durations of 3-5 ms. C: IK during a prolonged depolarization to +40 mV inactivates similarly in both cases. D-G: population analysis of voltage-dependent gating properties in oocytes expressing SqKv1A Delta V5 G87R (filled circles) and SqKv1D (open circles). Means ± 1 SE are plotted (n = 3). No differences between data from two channel types are significant by t-test. D: peak gK: gK from each patch was estimated as Delta I/Delta V after repolarizing to -80 mV at the time of peak IK (see inset and METHODS) and normalized by gKmax, the maximal value for that patch. E: activation kinetics: tau ON was estimated by fitting a single exponential to IK during the final 40-50% approach to peak IK. F: deactivation kinetics: tau OFF was estimated by fitting a single exponential to the tail IK following a 3-5 ms pulse to +40 mV. G: inactivation kinetics: tau INAC was estimated by fitting a single exponential to the decay of IK during a 500-ms pulse. All of the above analyses were also conducted for SqkV1B G36S. No significant differences in properties were found from those of the above two channel types (data are not illustrated).

Identification of a residue in the T1 domain of SqKv1B that impairs expression

SqKv1B lacks the expression-inhibiting elements of SqKv1A discussed above. It has the consensus Arg in FFDR (at the site equivalent to G87 of SqKv1A) and lacks Val 5 and the other first 11 amino acids comprising the extreme N-terminus of SqKv1A. As indicated in Fig. 2, however, SqKv1B still expresses at a much lower level than does SqKv1D. There are only two amino acid differences in the entire N-terminal and T1 regions of the latter two isoforms (Fig. 1), and one of these (corresponding to K132 in SqKv1A) is not highly conserved among Kv1 members. However, at the site of S47 in SqKv1A, a consensus Ser exists in all Kv1 members except SqKv1B which has Gly.

Changing this anomalous residue of SqKv1B to the consensus Ser (SqKv1B G36S) increases the level of functional expression ~10-fold to the level shown by SqKv1D (Fig. 6A) with no significant effect on voltage-dependent properties of IK as determined in two-microelectrode recordings (Fig. 6, B and C). Cell-attached macropatch experiments confirmed that properties of IK for G36S channels are indistinguishable from those analyzed in conjunction with Fig. 5 for the other two high-expression variants (data not illustrated). The converse manipulation was carried out using SqKv1D, i.e., the consensus Ser at the equivalent position was changed to Gly (SqKv1D S13G). This mutation leads to a large decrease in the level of functional expression (Fig. 6A) without significantly affecting voltage-dependent properties determined by two-electrode voltage clamp (Fig. 6, D and E). Unfortunately the low level of expression preludes high-quality macropatch analysis. Thus, the identity of this single amino acid appears to completely account for the difference in expression levels shown by SqKv1B and D. 



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Fig. 6. Gly substitution for Ser at the T1 consensus SGLR reduces functional expression in SqKv1B and SqKv1D. A: mean IK at +10 mV (±SE; n = 5) are plotted for SqKv1B, SqKv1B G36S, SqKv1D, and SqKv1D S13G. Replacement of the anomalous Gly36 with Ser in SqKv1B increases the level of functional expression ~10-fold. Glycine replacement of the consensus Ser13 in SqKv1D causes an approximately reciprocal decrease in functional expression. B-E: representative IK traces (indicated voltages apply to each panel) for these 4 channel types reveal no large alteration in voltage-dependent properties due to the SqKv1B G36S and SqKv1D S13G mutations. Scale bars apply throughout.

Anomalous T1 residues also affect the level of alpha -subunit protein

Conventional immunoblotting methods were used to investigate the effects on the total level of alpha -subunit protein produced by the manipulations of SqKv1A and B that increased surface expression of functional K channels. The crude membrane fraction used for these experiments contains material both from surface and internal membrane pools, and analysis of the glycosylation state of bands detected is presented below.

SQKV1A PROTEIN: EFFECTS OF THE G87R AND Delta V5 MUTATIONS. Increased functional expression due to both manipulations of SqKv1A discussed above is accompanied by a large increase in total alpha -subunit protein. Immunoblots were prepared from equal numbers of oocytes injected with cRNA for either wild-type SqKv1A or SqKv1A G87R, and typical results are illustrated in Fig. 7A. The single band detected with an apparent molecular weight of about 52 kDa represents core-glycosylated protein (as demonstrated below) and is much more prevalent when the native Gly (G87 lane) is replaced by Arg (R87 lane). Introduction of the same G87R mutation into the background of SqKv1A Delta V5 leads to a similar large increase in the intensity of the 52-kDa band, and multiple bands of higher apparent molecular weight become very prominent (Fig. 7B). As demonstrated below, these later bands represent glycosylated alpha -subunits in which carbohydrate has been additionally processed. These higher molecular weight bands are more prevalent with those constructs that support a high level of functional expression.



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Fig. 7. Influence of the anomalous Gly residues in conserved T1 sequences (FFDR right-arrow G and S right-arrow GGLR) on expression of SqKv1 alpha -subunit protein. Immunoblots using affinity-purified SqKv1-specific antibodies were carried out 3 days postinjection. Five oocytes for each indicated cRNA were processed (see METHODS) and loaded in each lane. In each panel A-E, the left lane represents the wild-type residue at the noted position of the construct, and the right lane represents the relevant mutation. A: restoration of the consensus FFDR87 in SqKv1A increases the amount of protein in the single band of ~52 kDa apparent molecular weight. As discussed in the text, this band represents core-glycosylated material. B: in the presence of the SqKv1A Delta V5 background, the G87R mutation increases the amount of core-glycosylated protein and reveals higher molecular-weight processed forms. C: both core-glycosylated and higher molecular weight bands are detectable for wild-type SqKv1D, and disruption of the consensus FFDR53 by the R53G mutation greatly decreases the amount of both forms. D: restoration of the consensus S36GLR in SqKv1B G36S causes a reduction in the amount of the core-glycosylated protein but an increase in higher molecular bands. E: disruption of the consensus G13GLR in SqKv1D S13G produces a large increase in the amount of core-glycosylated protein.

The position equivalent to G87 was also investigated in SqKv1D (R53). Core-glycosylated and higher molecular weight bands are generally detectable in SqKv1D samples (Fig. 7C, R53 lane), but replacement of the consensus Arg with Gly (SqKv1D R53G) eliminates the upper bands and reduces the intensity of the core-glycosylated band (Fig. 7C, G53 lane). In addition, functional expression appears to be abolished in SqKv1D R53G (not illustrated). Thus, both the physiological and biochemical results are complementary to those described above for the opposite mutation in SqKv1A.

SQKV1B PROTEIN: EFFECTS OF THE G36S MUTATION. Immunoblots of SqKv1B samples typically show a large amount of a single, core-glycosylated band with little or no detectable higher molecular weight material. Introduction of the G36S mutation, which greatly increases functional expression, is accompanied by a reduction in intensity of the band of lower apparent molecular weight and enhancement of higher molecular weight forms (Fig. 7D). The reverse mutation made in SqKv1D at the analogous position (SqKv1D S13G) results in a dramatic increase in intensity of the lower molecular weight band (Fig. 7E). This mutation also greatly decreases functional expression (Fig. 4).

Glycosylation of SqKv1 alpha -subunits

The strong correlation between the level of functional K-channel expression and the prominence of higher molecular weight alpha -subunit protein described above for the three SqKv1 isoforms suggests that the identified deviations from the T1 consensus may affect both assays by a common mechanism. Here we analyze the glycosylation state of SqKv1A protein and make use of this information to judge where in the biosynthetic pathway the anomalous T1 residues exert their deleterious effects on expression.

IDENTIFICATION OF CORE-GLYCOSYLATED AND MORE HIGHLY PROCESSED FORMS OF SQKV1A alpha -SUBUNITS. N-linked glycosylation of membrane proteins occurs cotranslationally in the ER where carbohydrate with a high mannose content is covalently linked to asparagine residues of the core polypeptide. These core-glycosylated polypeptides are then transported to the Golgi apparatus where the core-carbohydrate trees are trimmed and processed before delivery of the mature protein to the surface membrane.

Tunicamycin prevents core-glycosylation, and culture of oocytes expressing wild-type SqKv1A in the presence of this agent results in a clear reduction of the apparent molecular weight of the single 52-kDa band detected (Fig. 8A; solid vs. open arrow). This suggests that the 52-kDa band represents a glycosylated form of the protein. Analyses carried out with SqKv1B and D isoforms revealed the same effect of tunicamycin (data not illustrated), and this indicates that the prominent single band detectable in these variants is also glycosylated. A similar shift of the 52-kDa band also occurs with SqKv1A Delta V5 G87R (Fig. 8B), but in this case the prominent bands in the 60-70 kDa range (bracket) are eliminated. This latter material thus appears to represent glycosylated alpha -subunits which have experienced additional carbohydrate processing in the Golgi.



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Fig. 8. Identification of glycosylated forms of SqKv1A alpha -subunits. A: effect of tunicamycin (TM) treatment (2 µg/ml; see METHODS) on expression of wild-type SqKv1A protein as determined by immunoblot analysis. In the absence of TM (- lane), the single detectable band has an apparent molecular weight of ~ 52 kD (filled arrow). Treatment with TM (+ lane) causes a downward shift of this band (hollow arrow). B: effect of TM treatment on expression of SqKv1A Delta V5 G87R. Control conditions (- lane) give rise to prominent bands of higher molecular weight (bracket) as well as the 52-kDa form (solid arrow). TM treatment (+ lane) eliminates the higher molecular weight forms and shifts the 52-kDa band downward (open arrow). C: endoglycosidase H (endo H) digestion of SqKv1A protein. HA-tagged SqKv1A Delta V5 G87R was expressed and immunoprecipitated with monoclonal anti-HA and subjected to digestion with endo H (see METHODS). Digestion produces a downward shift of the prominent lower band (solid arrow in -endo H lane versus hollow arrow in +endo H lane) similar to that produced by TM treatment. Although higher molecular weight forms of the protein are not well resolved, endo-H digestion does not greatly alter the appearance of this material. Control lane represents immunoprecipitated proteins from labeled oocytes that were not injected with cRNA.

Identification of the 52-kDa band as the core-glycosylated form was also made by assessing the effects of digestion of SqKv1A protein with endoglycosidase H (endo H), an enzyme which specifically cleaves the high-mannose oligosaccharides that are attached in the ER and later removed in the Golgi. Sensitivity to endo H digestion identifies core-glycosylated polypeptides, whereas resistance identifies more fully processed protein.

Immunoprecipitation of metabolically labeled SqKv1A Delta V5 G87R carrying an HA-epitope tag was carried out from crude membranes using a monoclonal HA antibody. After digestion with endo H, this material was analyzed using SDS-PAGE and autoradiography. Results in Fig. 8C indicate that the band corresponding to the 52-kDa form is sensitive to endo H digestion (solid vs. open arrows) and that the decrease in apparent molecular weight is the same as that produced by tunicamycin treatment. Thus, this band represents core-glycosylated protein that has not yet been processed in the Golgi. Material in the 60-70 kDa range (bracket) appears to be resistant to endo-H treatment, consistent with the idea that this material represents more complex and fully processed glycosylated forms.

Attempts to carry out the same endo-H analysis with the poorly expressing variants SqKv1B and SqKv1D S13G were not successful, presumably due to the lower amount of total protein produced. Tunicamycin treatment reduces the apparent molecular weight of these alpha -subunits to the same degree as with SqKv1A Delta V5 G87R, however. This strongly suggests that most SqKv1B and D protein has been core-glycosylated but has experienced little additional processing.

IDENTIFICATION OF N-LINKED GLYCOSYLATION SITES. Three extracellular consensus sites for N-linked glycosylation exist in each of the natural SqKv1 isoforms, and these are located in the S1-S2 and S3-S4 linkers (Fig. 9A; numbering for SqKv1A). To determine which of these sites were actually glycosylated in Xenopus oocytes, we used site-directed mutagenesis to alter them individually in the background of SqKv1A Delta V5 G87R (N202Q, N272Q, and N281Q). In the case of the S1-S2 mutant, all signs of glycosylation are eliminated (N202Q lane in Fig. 9B). The more fully processed bands are absent, and the 52-kDa core-glycosylated band is shifted downward to the same extent as that produced by tunicamycin treatment (Fig. 7, A and B). Tunicamycin treatment of oocytes expressing this N202Q mutant produced no additional decrease in apparent molecular weight (data not illustrated). Mutations at the sites in the S3-S4 linker (N272Q and N281Q) did not significantly alter the banding patterns in Western blots (Fig. 9B).



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Fig. 9. Identification of an N-linked glycosylation site in SqKv1A. A: schematic of an SqKv1A alpha -subunit showing the location of potential N-linked glycosylation sites (Asn-X-Thr/Ser consensus). B: immunoblot analysis of oocytes expressing either control channels (SqKv1A Delta V5 G87R) or the individual mutations indicated for each lane. Elimination of higher molecular weight forms (bracket) and a downward shift of the 52-kDa band (solid arrow), consistent with a total lack of glycosylation, are evident only for the N202Q mutation. No detectable changes occur with mutations at the other sites.

Thus, it appears that SqKv1A is glycosylated only at a single site (N202) in the S1-S2 linker. Similar results were obtained with the analogous mutations in SqKv1B G36S and in SqKv1D. In both cases, only the S1-S2 site was glycosylated (N191Q in SqKv1B and N168Q in SqKv1D; data not illustrated).

EFFECTS OF GLYCOSYLATION ON FUNCTIONAL EXPRESSION. Oocytes expressing the glycosylation mutants discussed above all produced functional K channels. An analysis of cell-attached patch data like that described in conjunction with Fig. 5 was carried out for the N202Q mutation of SqKv1A Delta V5 G87R. Voltage-dependent properties of macroscopic IK were no different from those in control oocytes (data not illustrated), and nonglycosylated channels SqKv1A channels therefore appear to function normally. Although a voltage shift in the gK-V relation has been reported for nonglycosylated mammalian Kv1.1 channels (Thornhill et al. 1996), this effect is not detected with SqKv1A or Shaker (Santacruz-Toloza et al. 1994).

Elimination of glycosylation did not significantly alter the level of functional expression (assayed with two-electrode voltage clamp) at times later than 1 day postinjection. Similar results have been reported for Shaker (Santacruz-Toloza et al. 1994) and mammalian Kv1.1 (Deal et al. 1994).

Effects of deglycosylation were also examined in conjunction with the rate at which functional SqKv1A channels appeared on the surface membrane following cRNA injection. The time course of gK development in oocytes expressing SqKv1A Delta V5 G87R with or without the N202Q mutation is illustrated in Fig. 10A. Mean gK values of the two data sets are not significantly different after 12 h postinjection, but at 6 h, the difference is highly significant (t-test; P < 0.007). At this early time, control oocytes expressed nearly six times as many functional channels as did oocytes expressing nonglycosylated channels. Similar differences between very early and later time points were observed in oocytes injected with cRNAs for SqKv1D or SqKv1B G36S and the respective mutants (SqKv1B N191Q and SqKv1D N168Q) in which glycosylation is abolished (data not illustrated).



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Fig. 10. Effect of glycosylation on the time course of functional expression of SqKv1A channels. gK was measured from individual eggs at various times after cRNA (2 ng) injection. A: mean gK (±SE; n = 5) in control oocytes expressing SqKv1A Delta V5 G87R channels () develops more rapidly than that in oocytes expressing glycosylation-deficient channels (N202Q mutation; open circle ). This effect is not detectable for points after 6 h. All data were obtained in the same batch of oocytes. B: gK values were determined in individual oocytes expressing control (SqKv1A Delta V5 G87R; ) or nonglycosylated (N202Q; open circle ) channels during the first 10 h postinjection. The rate of appearance of gK is substantially reduced for by the N202Q mutation. Lines were fit by eye.

The delayed appearance of functional nonglycosylated SqKv1A Delta V5 G87R channels was examined more closely in another set of experiments in which gK was determined for individual oocytes starting 30 min postinjection (Fig. 9B). IK was first detectable at 2.5 h for control glycosylated channels but not until after 4.5 h for nonglycosylated channels. Furthermore, the initial rate of gK appearance is lower for the glycosylation mutants (14.4 µS/h vs. 46.3 µS/h for controls). Thus, glycosylated SqKv1A channels appear on the surface membrane with a smaller delay and at a higher initial rate than do their nonglycosylated counterparts.


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Control of Kv channel density in neurons and other cells may involve regulation at the level of protein synthesis, processing, trafficking, or degradation. The general sequence of events in the biochemical maturation of mammalian (Deal et al. 1994) and insect (Nagaya and Papazian 1997; Schulteis et al. 1998) Kv1 channels has been determined in heterologous expression systems. N-linked glycosylation occurs during translation, and assembly of core-glycosylated alpha -subunits into tetramers occurs in the ER. Complex processing of carbohydrate, additional folding of the mature channel, and sorting for delivery to the surface membrane take place later in the Golgi. These basic features presumably also apply to squid Kv1 channels.

Impaired expression of SqKv1A and B may result from defects early in biosynthesis

Aberrant amino acids in two extremely conserved segments of the T1 domain in SqKv1A (FFDG87 vs. FDDR, Fig. 1B) and SqKv1B (G36GLR vs. SGLR, Fig. 1B) greatly inhibit functional expression of the wild-type channels in Xenopus oocytes (Figs. 3, 4, and 6). The low level of gK observed reflects a reduced density of functionally normal K channels and is not due to changes in unitary conductance or gating properties.

Immunoblot analyses reveal that fully mature, glycosylated protein is greatly reduced by glycine in either of the above positions and that only core-glycosylated alpha -subunits are detectable. This suggests that very little wild-type protein moves from the ER to the Golgi for final processing and ultimately to the surface membrane where functional channels can be detected. The aberrant glycine residues therefore appear to exert their influence early in biosynthesis, most likely in the ER, where core-glycosylated subunits probably accumulate until being degraded. Mechanisms leading to the proposed failure of wild-type SqKv1A and B to efficiently exit the ER merit further investigation.

Effects of glycine at these two sites on the level of core-glycosylated protein are not the same, however. In SqKv1A, the FFDG87 anomaly reduces the level of core-glycosylated protein (Fig. 7, A-C), whereas the level is increased by G36GLR in SqKv1B (Fig. 7, D and E). This discrepancy may reflect different degradation rates for each type of defective alpha -subunit.

Structural implications of the aberrant T1 residues in SqKv1A and B

Exactly how the T1 domain confers sub-family specificity to tetramerization of Kv channels is not well understood, but the positions studied here in SqKv1A (FFDG87) and SqKv1B (G13GLR) correspond to structurally important points identified in the tetrameric T1 complex of an Aplysia Kv1 member (Bixby et al. 1999; Kreusch et al. 1998), the equivalent Aplysia positions being FFDR113 and S73GLR. Based on this structure, it is likely that glycine substitution at these positions generates different structural defects.

In the first case, the FFDR113 site (Aplysia nomenclature throughout) occupies a hinge-like position in a linker connecting well-defined layers in the T1 tertiary structure, and interaction of R113 is predicted with Gln126 on a neighboring alpha -subunit (His in SqKv1A-D). Although the functional importance of R113 has not been determined, substitution of Gly for Arg at this position might disrupt overall structural integrity to a point that could compromise oligomerization.

The second site in Aplysia, S73GLR, has been more thoroughly studied, and structural analysis indicates that it interacts with both Gln126 and Glu78 (conserved in SqKv1s). Substitution of Ser with Gly at this site greatly impedes binding of the mutant and wild-type T1 proteins (Bixby et al. 1999), and this residue thus appears to be important in conferring sub-family specificity to tetramerization of Kv1 alpha -subunits. Self-association of G73GLR constructs was not tested in the cited study, but inefficient tetramerization due to this defect would be consistent with observations presented in the present paper.

Impaired functional expression due to distal N-terminal elements in SqKv1A

Deletion of first 34 amino acids of SqKv1A, or even of just Val at position 5, results in a large increase in functional channel density (Fig. 3D). Neither mutation alters other functional channel properties, and both increase the total amount of channel protein accumulated (Figs. 7, A and B, and 8, A and B).

Mechanisms underlying these effects are not clear. One possibility stems from the similarity of the first 12 amino acids of SqKv1A to conventional leader sequences that are cleaved in the ER during biosynthesis of many membrane and secretory proteins (Dalbey and von Heijne 1992; von Heijne 1990). Although the N-terminus of SqKv1A is recognized as a signal-peptide (Nielsen et al. 1997; www.cbs.dtu.dk/services/SignalP/), the region upstream of the predicted cleavage site (15 amino acids) is unusually short and may therefore not serve as a very good leader. Nonetheless, the N-terminus of SqKv1A might interact with the oocyte ER membrane in an abnormal way and perturb the conformation of nascent alpha -subunits. Such defective subunits would probably be rapidly degraded. Although functional channels can form from true wild-type SqKv1A subunits, this appears to occur with extremely low efficiency.

A vaguely defined "signal sequence" located in the first 34 amino acids of a rabbit Kv1.3 alpha -subunit also inhibits expression in oocytes through an undefined mechanism (Segal et al. 1999; Yao et al. 1998). Although the extreme N-terminus of Kv1.3 is rather hydrophobic, it is clearly not identified as a signal peptide by the algorithm cited above. Furthermore, deletion of amino acids 3-27 of Kv1.3 did not increase expression, and an inhibitory mechanism of the type postulated for SqKv1A therefore seems unlikely.

Functional roles for glycosylation of SqKv1 channels

Kv1 alpha -subunits from squid (Fig. 9), insects (Santacruz-Toloza et al. 1994), and mammals (Deal et al. 1994; Shi et al. 1996) are all glycosylated within a stretch of nine amino acids in the middle of the S1-S2 linker, a region that shows no primary sequence similarity, even between mammalian Kv1s (Stühmer et al. 1989). Although the common location of this glycosylation site suggests that carbohydrate attachment here is important, the functional significance of this modification remains obscure.

A novel finding in this paper concerns the increased delay and decreased rate in the appearance of functional, nonglycosylated SqKv1 channels in the oocyte membrane following cRNA injection (Fig. 10B). At times later than 12 h postinjection, however, this inhibitory effect is no longer detectable (Fig. 10A), and gK rises at a similar rate to a comparable final level for both normal and nonglycosylated channels.

These kinetic manifestations suggest a functional role for glycosylation. The significance of the abnormally long delay in the appearance of functional, nonglycosylated channels after cRNA injection can be viewed in the same manner as the delay in IK activation after a voltage-clamp step. Sigmoidal IK kinetics indicates multiple closed states in the activation pathway. Similarly, the delayed appearance of nonglycosylated channels indicates that they have either passed through more steps during processing or through the same steps more slowly than their glycosylated counterparts do.

This analogy can also be extended to later times. Rate-limiting steps in channel activation act to determine the limiting "final" time course at which IK develops following a voltage step. Rates of appearance for glycosylated and nonglycosylated channels are identical more than 12 h postinjection, suggesting that the rate-limiting steps in channel processing are not glycosylation sensitive.

The gating analogy would suggest that the glycosylation-sensitive steps precede the rate-limiting step, and this would be consistent with an involvement of glycoprotein-specific chaperones, like calnexin (Nagaya et al. 1999). Transient interaction between calnexin and glycosylated Shaker protein occurs early in biosynthesis in the ER. The functional importance of this interaction is not known for Kv1 proteins, but calnexin-binding might increase the efficiency of early processing steps or act to bypass certain steps. In either case, nonglycosylated channels, which do not interact with calnexin, would move through the ER more slowly. If a subsequent step, e.g., sorting in the Golgi or delivery to the surface membrane, was rate-limiting, the time course for the appearance of functional channels would be affected by the lack of glycosylation in the way observed for SqKv1A.

Expression of SqKv1 channels in the squid nervous system

Experiments described in this paper were carried out on cloned squid channels expressed in a heterologous expression system. How applicable are these results to natural expression of the corresponding channels in GFL neurons (SqKv1A) and other neurons of the stellate ganglion (SqKv1B and D)? Functional expression of SqKv1B and D channels in oocytes is inversely proportional to the level of mRNAs for these species detected in the stellate ganglion with in situ hybridizations (Rosenthal et al. 1997). The functional significance of this correlation is unclear. It is not known whether these two isoforms are expressed in individual neurons, but the probability of heteromultimer formation would appear to be greatly reduced by the anomalous Gly36 of SqKv1B based on data discussed above (Bixby et al. 1999).

In the case of SqKv1A, which is only expressed in GFL neurons, two major questions arise. The first concerns the putative sequence peptide in the N-terminus. How this unusual feature impacts functional expression in vivo is unknown and merits study. The second question involves the FFDG87 anomaly in T1. Preliminary evidence suggests that editing of SqKv1A mRNA occurs at this position in vivo (Rosenthal and Bezanilla 2000). How the resulting glycine substitution at a universally conserved Kv residue affects SqKv1A expression in GFL neurons, and how mRNA editing might be involved in controlling channel density in GFL neurons remain outstanding questions.


    ACKNOWLEDGMENTS

We posthumously thank J. Kono for many years of support. This work constituted part of T. Liu's Ph.D. thesis from Stanford University.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-17510 to W. F. Gilly.

Present address of J.J.C. Rosenthal: Dept. of Physiology, UCLA School of Medicine, Los Angeles, CA 90095.


    FOOTNOTES

Address for reprint requests: W. F. Gilly, Hopkins Marine Station, Pacific Grove, CA 93950 (E-mail: lignje{at}leland.stanford.edu).

Received 8 May 2000; accepted in final form 15 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
Methods
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society