A family of delayed rectifier Kv1 cDNAs showing cell type-specific expression in the squid stellate ganglion/giant fiber lobe complex

Abstract

Squid giant axons are formed by giant fiber lobe (GFL) neurons of the stellate ganglion (SG). Other large motoneurons in the SG form a parallel system. A small family of cDNAs (SqKv1A-D) encoding Kv1 alpha-subunits was identified in a squid (Loligo opalescens) SG/GFL library. Members have distinct 5' untranslated regions (UTRs) and initial coding regions, but beyond a certain point (nucleotide 34 of SqKv1A) only nine differences exist. 3' UTRs are identical. Predicted alpha-subunits are nearly identical, and only the N termini differ significantly, primarily in length. RNase protection assays that use RNA isolated from specific SG regions show that SqKv1A mRNA is expressed prominently in the GFL but not in the SG proper. SqKv1B yields the opposite pattern. SqKv1D also is expressed only in the SG. SqKv1C expression was not detectable. In situ hybridizations confirm these results and reveal that SqKv1B mRNA is abundant in many large neurons of the SG, whereas SqKv1D expression is limited to small isolated clusters of neurons. SqKv1A and B are thus the predominant Kv1 mRNAs in the SG/GFL complex. Activation properties of SqKv1A and B channels expressed in oocytes are very similar to one another and compare favorably with properties of native delayed rectifier channels in GFL neurons and large SG neurons. The Kv1 complement in these squid neurons thus seems to be relatively simple. Several differences exist between cloned and native channels, however, and may reflect differences in the cellular environments of oocytes and neurons.

Fig. 1.

Structure of the SqKv1 cDNAs. A, Schematic of SqKv1A coding and UTRs (open andsolid segments, respectively) showing the locations of oligonucleotides used in this study (see Table 1) and of the probe (pSKC1; Rosenthal et al., 1996) used to screen the cDNA library. Cross-hatched segments represent transmembrane regions S1–S6. The restriction sites indicated are discussed in Materials and Methods and Results.B, Nucleotide sequences of SqKv1A–D cDNAs.Numbering pertains to SqKv1A. Identities to SqKv1A are indicated by dashes. Start codons (boldface and underlined) were assigned as the first Met codon that yielded the longest open reading frame. Stop codons are boxed. The four nucleotide sequences become virtually identical downstream from position 35 of SqKv1A. The locations of the NdeI and EagI sites discussed in Materials and Methods and Results are indicated forSqKv1A. The complete 5′ UTR sequence is given forSqKv1D; the others have been truncated arbitrarily. Complete sequences will be deposited in GenBank on acceptance of this manuscript.

Fig. 2.

Differences in predicted primary structure of squid Kv1 α-subunits, based on SqKv1A–D cDNAs. A, Alignment of the predicted N-terminal portions of SqKv1A–D. Identical residues are boxed. Illustrated sequences stop at the position equivalent to G48 (diamond) of SqKv1A because all four of the predicted SqKv1 channels are identical after this point, with the exception of the four sites noted in B. The complete sequence of SqKv1A is given elsewhere (Rosenthal et al., 1996). A consensus intracellular PKC site (asterisk) is indicated near the start of the NAB domain (indicated by white bar below the sequences). B, Schematic of the SqKv1A protein between G48 (diamond) and the C terminus. The four residues downstream from G48 that differ in SqKv1A–D are noted, and their approximate locations are indicated. The white segment of the channel corresponds to the continuation of the NAB domain. Consensus intracellular PKC sites (asterisks) and extracellular N-linked glycosylation sites (filled circles) are indicated. SqKv1A–D show no consensus PKA sites.

Fig. 3.

Expression of SqKv1A, B, and D mRNAs in the SG/GFL complex, as determined by RNase protection assays. Predicted sizes for undigested probes and fully protected bands are (in nt)234/204 (SqKv1A), 404/347 (SqKv1B), and215/175 (SqKv1D). A, RNase protection assays were performed with SqKv1A probe (Probe A) and RNA selectively isolated from either SG and GFL, as indicated at thetop of each lane. GFL, Giant fiber lobe;SG, anterior portion of stellate ganglion (see Materials and Methods). tRNA served as a negative control. Nondigested probe provided a size marker. A fully protected band is prominent in the GFL lane (7 d exposure).B, RNase protection assays were performed with SqKv1B probe (Probe B) in the manner described. A prominent, fully protected band is evident in the SG lane (7 d exposure). C, Results of another experiment in which assays were performed with both SqKv1A and B probes (Probe A/B Mixed). A 204 nt band corresponding to SqKv1A mRNA is very prominent in the GFL lane; a 347 nt band corresponding to SqKv1B is apparent in the SG lane. D, RNase protection assays performed with SqKv1D probe (Probe D) yield a clear band only in the SG lane (1 d exposure; same experimental series as A,B). See text for additional details.

Fig. 4.

Selective expression patterns of SqKv1A, B, and D mRNAs in the SG/GFL complex. Ai, Horizontal section of a stellate ganglion stained with basic fuchsin and toluidine blue. The GFL lies on the left (posterior), and the SG proper is on the right surrounding the large pale neuropil region. Stellar nerves project laterally (bottom), and two giant axons are indicated (asterisks). Tracts of fusing GFL axons are indicated by arrowheads. Scale bar equals 0.5 mm in Ai, Bi, Ci, andDi and 0.1 mm in Aii, Bii,Cii, Dii, and E.Aii, Higher magnification of the boxed region in Ai illustrating the boundary between GFL neurons and the much larger SG neurons in the bottom right corner. Bi, In situ hybridization of ³⁵S-labeled cRNA probe specific for SqKv1A detects signal only from GFL neurons.Bii, Higher magnification of the boxed region in Bi enclosing the SG/GFL boundary. Virtually no cells are labeled in the SG proper, although a very small number of cells near the boundary seem to express SqKv1A. One such cell is visible in the bottom right corner.Ci, In situ hybridization of³⁵S-labeled cRNA probe specific for SqKv1B detects signal almost exclusively in SG neurons. Cii, Higher magnification of the boxed region in Cienclosing the SG/GFL boundary. Several very large SG neurons are heavily labeled. Di, Control in situhybridization of ³⁵S-labeled cRNA sense probe.Dii, Higher magnification of the boxed region in Di. This region of the SG proper includes neuropil (bottom right) and tracts of small axons projecting from the GFL into the SG (top).E, In situ hybridization of³⁵S-labeled cRNA probe specific for SqKv1D of a SG region equivalent to that in Dii. Tracts of GFL axons are at the top. A cluster of fairly large neurons in thecenter of the picture is labeled above background and lies just below these axons.

Fig. 5.

Activation properties of macroscopicI_K recorded from oocytes and squid neurons. Cell-attached patch currents are illustrated at the indicated voltages from oocytes injected with cRNA for SqKv1A (A) or SqKv1B (B) and from cell bodies of GFL neurons (C) and large SG neurons (D). The general form ofI_K is similar in all cases.E, Comparison of voltage dependence ofg_K for cloned and native K channels. Relative g_K was estimated as ΔI/ΔV after repolarization at the time of peak I_K (see inset).Symbol code is indicated on the figure. Plottedpoints are mean values ± 1 SEM. The number of patches (n) is indicated, except wheren = 3 or 4. Error bars are omitted when smaller than the symbol or when n < 3. F, Activation kinetics (t_½) were assayed as the time to reach 50% peak I_K.Symbols are plotted as in A.

Fig. 6.

Deactivation properties of macroscopicI_K recorded from oocytes and squid neurons.A, Tail currents in a patch from an oocyte injected with SqKv1B cRNA were measured at the indicated voltages after a 25 msec test pulse (see inset). B, Analogous tail current records from a patch on a large SG neuron. Note the difference in time scale from A. C, Tail currents from a patch on a GFL neuron. D, Deactivation kinetics (τ_OFF) were obtained by fitting a single exponential to the tail current after repolarization of a brief test pulse (usually 25 msec duration). Symbols are plotted as in Figure 5.