The sodium channel beta-subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart.

1. Cardiac sodium channels are composed of a pore-forming alpha-subunit, SCN5a, and one or more auxiliary beta-subunits. The aim of this study was to investigate the role of the recently discovered member of the beta-subunit family, SCN3b, in the heart. 2. Northern blot and Western blot studies show that SCN3b is highly expressed in the ventricles and Purkinje fibres but not in atrial tissue. This is in contrast to the uniform expression of SCN1b throughout the heart. 3. Co-expression of SCN3b with the cardiac-specific alpha-subunit SCN5a in Xenopus oocytes resulted in a threefold increase in the level of functional sodium channel expression, similar to that observed when SCN1b was co-expressed with SCN5a. These results suggest that both SCN1b and SCN3b improve the efficiency with which the mature channel is targeted to the plasma membrane. 4. When measured in cell-attached oocyte macropatches, SCN3b caused a significant depolarising shift in the half-voltage of steady-state inactivation compared to SCN5a alone or SCN5a + SCN1b. The half-voltage of steady-state activation was not significantly different between SCN5a alone and SCN5a + SCN3b or SCN5a + SCN1b. 5. The rates of inactivation for SCN5a co-expressed with either subunit were not significantly different from that for SCN5a alone. However, recovery from inactivation at -90 mV was significantly faster for SCN5a + SCN1b compared to SCN5a + SCN3b, and both were significantly faster than SCN5a alone. 6. Thus, SCN1b and SCN3b have distinctive effects on the kinetics of activation and inactivation, which, in combination with the different patterns of expression of SCN3b and SCN1b, could have important consequences for the integrated electrical activity of the intact heart.

Developmental expression of the novel voltage-gated sodium channel auxiliary subunit beta3, in rat CNS.

1. We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Nabeta1.1 and Nabeta2.1 and the novel, recently cloned subunit, beta3. 2. In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and beta3 mRNA at embryonic stages whilst Nabeta1.1 and Nabeta2.1 mRNA was absent throughout this period. 3. Nabeta1.1 and Nabeta2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. beta3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum. 4. Emulsion-dipped slides showed co-localisation of beta3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction. 5. Co-expression in Xenopus oocytes revealed that beta3 could modify the properties of Nav1.3; beta3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation. 6. In conclusion, beta3 is shown to be the predominant beta subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage-gated sodium channels during development.

Tissue distribution and functional expression of the human voltage-gated sodium channel beta3 subunit.

This study investigated the distribution of beta3 in human tissues and the functional effects of the human beta3 subunit on the gating properties of brain and skeletal muscle alpha subunits. Using RT-PCR of human cDNA panels, beta3 message was detected in brain, heart, kidney, lung, pancreas and skeletal muscle. Both alphaIIA and SkM1 expressed in Xenopus oocytes inactivated with a time course described by two exponential components representing fast and slow gating modes, while co-expression of human beta3 with alphaIIA or SkM1 significantly increased the proportion of channels operating by the fast gating mode. In the presence of beta3 a greater proportion of alphaIIA or SkM1 current was described by the fast time constant for both inactivation and recovery from inactivation. beta3 caused a hyperpolarizing shift in the voltage dependence of inactivation of alphaIIIA and reduced the slope factor. The voltage dependence of inactivation of SkM1 was described by a double Boltzmann equation. However, SkM1 co-expressed with beta3 was described by a single Boltzmann equation similar to one of the Boltzmann components for SkM1 expressed alone, with a small positive shift in V1/2 value and reduced slope factor. This is the first study demonstrating that beta3 is expressed in adult mammalian skeletal muscle and can functionally couple to the skeletal muscle alpha subunit, SkM1.

beta3, a novel auxiliary subunit for the voltage-gated sodium channel, is expressed preferentially in sensory neurons and is upregulated in the chronic constriction injury model of neuropathic pain.

Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage-gated sodium channel alpha-subunits. However, of the auxiliary subunits, beta1 is expressed preferentially in only large- and medium-diameter neurons of the DRG while beta2 is absent in all DRG cells. In view of this, we have compared the distribution of beta1 in rat DRG and spinal cord with a novel, recently cloned beta1-like subunit, beta3. In situ hybridization studies demonstrated high levels of beta3 mRNA in small-diameter c-fibres, while beta1 mRNA was virtually absent in these cell types but was expressed in 100% of large-diameter neurons. In the spinal cord, beta3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while beta1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of beta3 expression in DRG appears to correlate with the TTX-resistant voltage-gated sodium channel subunit PN3, we co-expressed the two subunits in Xenopus oocytes. In this system, beta3 caused a 5-mV hyperpolarizing shift in the threshold of activation of PN3, and a threefold increase in the peak current amplitude when compared with PN3 expressed alone. On the basis of these results, we examined the expression of beta-subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in beta3 mRNA expression in small-diameter sensory neurons of the ipsilateral DRG. These results show that beta3 is the dominant auxiliary sodium channel subunit in small-diameter neurons of the rat DRG and that it is significantly upregulated in a model of neuropathic pain.

beta 3: an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics.

The voltage-sensitive sodium channel confers electrical excitability on neurons, a fundamental property required for higher processes including cognition. The ion-conducting alpha-subunit of the channel is regulated by two known auxiliary subunits, beta1 and beta2. We have identified rat and human forms of an additional subunit, beta3. It is most closely related to beta1 and is the product of a separate gene localized to human chromosome 11q23.3. When expressed in Xenopus oocytes, beta3 inactivates sodium channel opening more slowly than beta1 does. Structural modeling has identified an amino acid residue in the putative alpha-subunit binding site of beta3 that may play a role in this difference. The expression of beta3 within the central nervous system differs significantly from beta1. Our results strongly suggest that beta3 performs a distinct neurophysiological function.

Bombesin-like peptides depolarize rat hippocampal interneurones through interaction with subtype 2 bombesin receptors.

1. Whole-cell patch-clamp recordings were made from visually identified hippocampal interneurones in slices of rat brain tissue in vitro. Bath application of the bombesin-like neuropeptides gastrin-releasing peptide (GRP) or neuromedin B (NMB) produced a large membrane depolarization that was blocked by pre-incubation with the subtype 2 bombesin (BB2) receptor antagonist [D-Phe6, Des-Met14]bombesin-(6-14)ethyl amide. 2. The inward current elicited by NMB or GRP was unaffected by K+ channel blockade with external Ba2+ or by replacement of potassium gluconate in the electrode solution with caesium acetate. 3. Replacement of external NaCl with Tris-HCl significantly reduced the magnitude of the GRP-induced current at -60 mV. In contrast, replacement of external NaCl with LiCl had no effect on the magnitude of this current. 4. Photorelease of caged GTPgammaS inside neurones irreversibly potentiated the GRP-induced current at -60 mV. Similarly, bath application of the phospholipase C (PLC) inhibitor U-73122 significantly reduced the size of the inward current induced by GRP. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single hippocampal interneurones demonstrated the expression of BB2 receptor mRNA together with glutamate decarboxylase (GAD67). 6. Although bath application of GRP or NMB had little or no effect on the resting membrane properties of CA1 pyramidal cells per se, these neuropeptides produced a dramatic increase in the number and amplitude of miniature inhibitory postsynaptic currents in these cells in a TTX-sensitive manner.

Bombesin receptors inhibit G protein-coupled inwardly rectifying K+ channels expressed in Xenopus oocytes through a protein kinase C-dependent pathway.

Although activation of G protein-coupled inward rectifying K+ (GIRK) channels by Gi/Go-coupled receptors has been shown to be important in postsynaptic inhibition in the central nervous system, there is also evidence to suggest that inhibition of GIRK channels by Gq-coupled receptors is involved in postsynaptic excitation. In the present study we addressed whether the Gq-coupled receptors of the bombesin family can couple to GIRK channels and examined the mechanism by which this process occurs. Different combinations of GIRK channel subunits (Kir3.1, Kir3.2, and Kir3.4) and bombesin receptors (BB1 and BB2) were expressed in Xenopus oocytes. In all combinations tested GIRK currents were reversibly inhibited upon application of the bombesin-related peptides, neuromedin B or gastrin-releasing peptide in a concentration-dependent manner. Incubation of oocytes in the phospholipase C inhibitor U73122 or the protein kinase C (PKC) inhibitors chelerythrine and staurosporine significantly reduced the inhibition of GIRK currents by neuromedin B, whereas the Ca2+ chelator, BAPTA-AM had no effect. The involvement of PKC was further demonstrated by direct inhibition of GIRK currents by the phorbol esters, phorbol-12,13-dibutyrate and phorbol-12-myristate-13-acetate. In contrast, the inactive phorbol ester 4alpha-phorbol and protein kinase A activators, forskolin and 8-bromo cAMP did not inhibit GIRK currents. At the single-channel level, direct activation of PKC using phorbol ester phorbol-12, 13-dibutyrate caused a dramatic reduction in open probability of GIRK channels due to an increase in duration of the interburst interval.

Identification of regions that regulate the expression and activity of G protein-gated inward rectifier K+ channels in Xenopus oocytes.

1. The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein-gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild-type subunits together with the m2 muscarinic receptor in Xenopus oocytes. 2. Co-expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1-Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane. 3. Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine-induced channel activity compared with the wild-type Kir3.2 homomeric channel. Cell-attached, single-channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings. 4. Individually substituting the N- and C-terminal tails of Kir3.1 for those of Kir3.2 showed that the C-terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N-terminal or core regions of this subunit. 5. The chimeric channel, 121, displayed a higher ratio of ACh-induced to basal activity than the Kir3.1-Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for G beta gamma. Our results suggest that substituting the Kir3.1 C-terminal tail for the Kir3.2 tail promotes the opening conformational change of the G beta gamma-bound channel. 6. The core and C-terminal regions of Kir3.1 independently conferred time dependence on voltage-dependent activation. The time constant (tau) was between 5 and 10 ms and varied little over the voltage range -60 to -120 mV.

Molecular determinants for assembly of G-protein-activated inwardly rectifying K+ channels.

Kir3.1 and Kir3.2 associate to form G-protein-activated, inwardly rectifying K+ channels. To identify regions involved in the coassembly of these subunits, truncated Kir3.1 polypeptides were coexpressed with epitope-tagged subunits in an in vitro translation system. N-terminal, C-terminal, and core region polypeptides were coimmunoprecipitated with both Kir3.2 and Kir3.1, suggesting that multiple elements distributed throughout the Kir3.1 polypeptide contribute to intersubunit binding interactions. The Kir3.2 C-terminal polypeptide coimmunoprecipitated with the Kir3.1 C-terminal polypeptide, but neither region recognized the N-terminal domain and core region of the Kir3.1 subunit. This suggests that within Kir3 channels the C-terminal domains of neighboring subunits interact. Coexpression of the truncated polypeptides with Kir3.1 and Kir3.2 in Xenopus oocytes reduced functional expression of the heteromeric channels. Constructs encoding the core region plus N-terminal and proximal C-terminal regions competed more effectively than the core region alone, which supports the contribution of all three regions to intersubunit binding interactions. Proximal and distal segments of the C-terminal domain were as effective at inhibiting functional expression as the entire C-terminal domain.

Talker continuity and the use of rate information during phonetic perception.

Research has shown that speaking rate provides an important context for the perception of certain acoustic properties of speech. For example, syllable duration, which varies as a function of speaking rate, has been shown to influence the perception of voice onset time (VOT) for syllable-initial stop consonants. The purpose of the present experiments was to examine the influence of syllable duration when the initial portion of the syllable was produced by one talker and the remainder of the syllable was produced by a different talker. A short-duration and a long-duration /bi/-/pi/ continuum were synthesized with pitch and formant values appropriate to a female talker. When presented to listeners for identification, these stimuli demonstrated the typical effect of syllable duration on the voicing boundary: a shorter VOT boundary for the short stimuli than for the long stimuli. An /i/ vowel, synthesized with pitch and formant values appropriate to a male talker, was added to the end of each of the short tokens, producing a new hybrid continuum. Although the overall syllable duration of the hybrid stimuli equaled the original long stimuli, they produced a VOT boundary similar to that for the short stimuli. In a second experiment, two new /i/ vowels were synthesized. One had a pitch appropriate to a female talker with formant values appropriate to a male talker; the other had a pitch appropriate to a male talker and formants appropriate to a female talker. These vowels were used to create two new hybrid continua. In a third experiment, new hybrid continua were created by using more extreme male formant values. The results of both experiments demonstrated that the hybrid tokens with a change in pitch acted like the short stimuli, whereas the tokens with a change in formants acted like the long stimuli. A fourth experiment demonstrated that listeners could hear a change in talker with both sets of hybrid tokens. These results indicate that continuity of pitch but not formant structure appears to be the critical factor in the calculation of speaking rate within a syllable.

Integrating speech information across talkers, gender, and sensory modality: female faces and male voices in the McGurk effect.

Studies of the McGurk effect have shown that when discrepant phonetic information is delivered to the auditory and visual modalities, the information is combined into a new percept not originally presented to either modality. In typical experiments, the auditory and visual speech signals are generated by the same talker. The present experiment examined whether a discrepancy in the gender of the talker between the auditory and visual signals would influence the magnitude of the McGurk effect. A male talker's voice was dubbed onto a videotape containing a female talker's face, and vice versa. The gender-incongruent videotapes were compared with gender-congruent videotapes, in which a male talker's voice was dubbed onto a male face and a female talker's voice was dubbed onto a female face. Even though there was a clear incompatibility in talker characteristics between the auditory and visual signals on the incongruent videotapes, the resulting magnitude of the McGurk effect was not significantly different for the incongruent as opposed to the congruent videotapes. The results indicate that the mechanism for integrating speech information from the auditory and the visual modalities is not disrupted by a gender incompatibility even when it is perceptually apparent. The findings are compatible with the theoretical notion that information about voice characteristics of the talker is extracted and used to normalize the speech signal at an early stage of phonetic processing, prior to the integration of the auditory and the visual information.