Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular KV currents and predisposes to ventricular arrhythmia.

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (KV) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)- and Brugada syndrome (BrS)-induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout ( Kcne5-/0) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5+/0 mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte KV current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current ( Ito,f). The current increases arose from an apex-specific increase in slow transient outward current-1 ( IKslow,1) (conducted by KV1.5) and Ito,f (conducted by KV4) and an increase in IKslow,2 (conducted by KV2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where KV2.1 was also detected in both Kcne5-/0 and Kcne5+/0 mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and KV2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases IK,slow2 by reducing KV2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of KV2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in KV2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of KV2.1 in mouse heart. Increased KV current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS.-David, J.-P., Lisewski, U., Crump, S. M., Jepps, T. A., Bocksteins, E., Wilck, N., Lossie, J., Roepke, T. K., Schmitt, N., Abbott, G. W. Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular KV currents and predisposes to ventricular arrhythmia.

Functional antagonism of voltage-gated K+ channel α-subunits in the developing brain ventricular system.

The brain ventricular system is essential for neurogenesis and brain homeostasis. Its neuroepithelial lining effects these functions, but the underlying molecular pathways remain to be understood. We found that the potassium channels expressed in neuroepithelial cells determine the formation of the ventricular system. The phenotype of a novel zebrafish mutant characterized by denudation of neuroepithelial lining of the ventricular system and hydrocephalus is mechanistically linked to Kcng4b, a homologue of the 'silent' voltage-gated potassium channel α-subunit Kv6.4. We demonstrated that Kcng4b modulates proliferation of cells lining the ventricular system and maintains their integrity. The gain of Kcng4b function reduces the size of brain ventricles. Electrophysiological studies suggest that Kcng4b mediates its effects via an antagonistic interaction with Kcnb1, the homologue of the electrically active delayed rectifier potassium channel subunit Kv2.1. Mutation of kcnb1 reduces the size of the ventricular system and its gain of function causes hydrocephalus, which is opposite to the function of Kcng4b. This demonstrates the dynamic interplay between potassium channel subunits in the neuroepithelium as a novel and crucial regulator of ventricular development in the vertebrate brain.

Targeted deletion of the Kv6.4 subunit causes male sterility due to disturbed spermiogenesis.

Electrically silent voltage-gated potassium (KvS) channel subunits (i.e. Kv5-Kv6 and Kv8-Kv9) do not form functional homotetrameric Kv channels, but co-assemble with Kv2 subunits, generating functional heterotetrameric Kv2--KvS channel complexes in which the KvS subunits modulate the Kv2 channel properties. Several KvS subunits are expressed in testis tissue but knowledge about their contribution to testis physiology is lacking. Here, we report that the targeted deletion of Kv6.4 in a transgenic mouse model (Kcng4-/-) causes male sterility as offspring from homozygous females were only obtained after mating with wild-type (WT) or heterozygous males. Semen quality analysis revealed that the sterility of the homozygous males was caused by a severe reduction in total sperm-cell count and the absence of motile spermatozoa in the semen. Furthermore, spermatozoa of homozygous mice showed an abnormal morphology characterised by a smaller head and a shorter tail compared with WT spermatozoa. Comparison of WT and Kcng4-/- testicular tissue indicated that this inability to produce (normal) spermatozoa was due to disturbed spermiogenesis. These results suggest that Kv6.4 subunits are involved in the regulation of the late stages of spermatogenesis, which makes them a potentially interesting pharmacological target for the development of non-hormonal male contraceptives.

Distinct Cell- and Layer-Specific Expression Patterns and Independent Regulation of Kv2 Channel Subtypes in Cortical Pyramidal Neurons.

The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K(+) current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT: Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.

Electrically silent Kv subunits: their molecular and functional characteristics.

Electrically silent voltage-gated potassium (KvS) α-subunits do not form homotetramers but heterotetramerize with Kv2 subunits, generating functional Kv2/KvS channel complexes in which the KvS subunits modulate the Kv2 current. This poses intriguing questions into the molecular mechanisms by which these KvS subunits cannot form functional homotetramers, why they only interact with Kv2 subunits, and how they modulate the Kv2 current.

Kv3 channels contribute to the delayed rectifier current in small cultured mouse dorsal root ganglion neurons.

Delayed rectifier voltage-gated K(+) (K(V)) channels are important determinants of neuronal excitability. However, the large number of K(V) subunits poses a major challenge to establish the molecular composition of the native neuronal K(+) currents. A large part (∼60%) of the delayed rectifier current (I(K)) in small mouse dorsal root ganglion (DRG) neurons has been shown to be carried by both homotetrameric K(V)2.1 and heterotetrameric channels of K(V)2 subunits with silent K(V) subunits (K(V)S), while a contribution of K(V)1 channels has also been demonstrated. Because K(V)3 subunits also generate delayed rectifier currents, we investigated the contribution of K(V)3 subunits to I(K) in small mouse DRG neurons. After stromatoxin (ScTx) pretreatment to block the K(V)2-containing component, application of 1 mM TEA caused significant additional block, indicating that the ScTx-insensitive part of I(K) could include K(V)1, K(V)3, and/or M-current channels (KCNQ2/3). Combining ScTx and dendrotoxin confirmed a relevant contribution of K(V)2 and K(V)2/K(V)S, and K(V)1 subunits to I(K) in small mouse DRG neurons. After application of these toxins, a significant TEA-sensitive current (∼19% of total I(K)) remained with biophysical properties that corresponded to those of K(V)3 currents obtained in expression systems. Using RT-PCR, we detected K(V)3.1-3 mRNA in DRG neurons. Furthermore, Western blot and immunocytochemistry using K(V)3.1-specific antibodies confirmed the presence of K(V)3.1 in cultured DRG neurons. These biophysical, pharmacological, and molecular results demonstrate a relevant contribution (∼19%) of K(V)3-containing channels to I(K) in small mouse DRG neurons, supporting a substantial role for K(V)3 subunits in these neurons.

Conserved negative charges in the N-terminal tetramerization domain mediate efficient assembly of Kv2.1 and Kv2.1/Kv6.4 channels.

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual alpha-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 alpha-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.

Independent movement of the voltage sensors in KV2.1/KV6.4 heterotetramers.

Heterotetramer voltage-gated K+ (KV) channels KV2.1/KV6.4 display a gating charge-voltage (QV) distribution composed by two separate components. We use state dependent chemical accessibility to cysteines substituted in either KV2.1 or KV6.4 to assess the voltage sensor movements of each subunit. By comparing the voltage dependences of chemical modification and gating charge displacement, here we show that each gating charge component corresponds to a specific subunit forming the heterotetramer. The voltage sensors from KV6.4 subunits move at more negative potentials than the voltage sensors belonging to KV2.1 subunits. These results indicate that the voltage sensors from the tetrameric channels move independently. In addition, our data shows that 75% of the total charge is attributed to KV2.1, while 25% to KV6.4. Thus, the most parsimonious model for KV2.1/KV6.4 channels' stoichiometry is 3:1.

Kv5, Kv6, Kv8, and Kv9 subunits: No simple silent bystanders.

Members of the electrically silent voltage-gated K(+) (Kv) subfamilies (Kv5, Kv6, Kv8, and Kv9, collectively identified as electrically silent voltage-gated K(+) channel [KvS] subunits) do not form functional homotetrameric channels but assemble with Kv2 subunits into heterotetrameric Kv2/KvS channels with unique biophysical properties. Unlike the ubiquitously expressed Kv2 subunits, KvS subunits show a more restricted expression. This raises the possibility that Kv2/KvS heterotetramers have tissue-specific functions, making them potential targets for the development of novel therapeutic strategies. Here, I provide an overview of the expression of KvS subunits in different tissues and discuss their proposed role in various physiological and pathophysiological processes. This overview demonstrates the importance of KvS subunits and Kv2/KvS heterotetramers in vivo and the importance of considering KvS subunits and Kv2/KvS heterotetramers in the development of novel treatments.

The contribution of Kv2.2-mediated currents decreases during the postnatal development of mouse dorsal root ganglion neurons.

Delayed rectifier voltage-gated K(+)(Kv) channels play an important role in the regulation of the electrophysiological properties of neurons. In mouse dorsal root ganglion (DRG) neurons, a large fraction of the delayed rectifier current is carried by both homotetrameric Kv2 channels and heterotetrameric channels consisting of Kv2 and silent Kv (KvS) subunits (i.e., Kv5-Kv6 and Kv8-Kv9). However, little is known about the contribution of Kv2-mediated currents during the postnatal development ofDRGneurons. Here, we report that the Stromatoxin-1 (ScTx)-sensitive fraction of the total outward K(+)current (IK) from mouseDRGneurons gradually decreased (~13%,P < 0.05) during the first month of postnatal development. Because ScTx inhibits both Kv2.1- and Kv2.2-mediated currents, this gradual decrease may reflect a decrease in currents containing either subunit. However, the fraction of Kv2.1 antibody-sensitive current that only reflects the Kv2.1-mediated currents remained constant during that same period. These results suggested that the fractional contribution of Kv2.2-mediated currents relative toIKdecreased with postnatal age. SemiquantitativeRT-PCRanalysis indicated that this decrease can be attributed to developmental changes in Kv2.2 expression as themRNAlevels of the Kv2.2 subunit decreased gradually between 1 and 4 weeks of age. In addition, we observed age-dependent fluctuations in themRNAlevels of the Kv6.3, Kv8.1, Kv9.1, and Kv9.3 subunits. These results support an important role of both Kv2 and KvS subunits in the postnatal maturation ofDRGneurons.

The anticonvulsant retigabine suppresses neuronal KV2-mediated currents.

Enhancement of neuronal M-currents, generated through KV7.2-KV7.5 channels, has gained much interest for its potential in developing treatments for hyperexcitability-related disorders such as epilepsy. Retigabine, a KV7 channel opener, has proven to be an effective anticonvulsant and has recently also gained attention due to its neuroprotective properties. In the present study, we found that the auxiliary KCNE2 subunit reduced the KV7.2-KV7.3 retigabine sensitivity approximately 5-fold. In addition, using both mammalian expression systems and cultured hippocampal neurons we determined that low µM retigabine concentrations had 'off-target' effects on KV2.1 channels which have recently been implicated in apoptosis. Clinical retigabine concentrations (0.3-3 µM) inhibited KV2.1 channel function upon prolonged exposure. The suppression of the KV2.1 conductance was only partially reversible. Our results identified KV2.1 as a new molecular target for retigabine, thus giving a potential explanation for retigabine's neuroprotective properties.

Modulation of Closed-State Inactivation in Kv2.1/Kv6.4 Heterotetramers as Mechanism for 4-AP Induced Potentiation.

The voltage-gated K+ (Kv) channel subunits Kv2.1 and Kv2.2 are expressed in almost every tissue. The diversity of Kv2 current is increased by interacting with the electrically silent Kv (KvS) subunits Kv5-Kv6 and Kv8-Kv9, into functional heterotetrameric Kv2/KvS channels. These Kv2/KvS channels possess unique biophysical properties and display a more tissue-specific expression pattern, making them more desirable pharmacological and therapeutic targets. However, little is known about the pharmacological properties of these heterotetrameric complexes. We demonstrate that Kv5.1, Kv8.1 and Kv9.3 currents were inhibited differently by the channel blocker 4-aminopyridine (4-AP) compared to Kv2.1 homotetramers. In contrast, Kv6.4 currents were potentiated by 4-AP while displaying moderately increased affinities for the channel pore blockers quinidine and flecainide. We found that the 4-AP induced potentiation of Kv6.4 currents was caused by modulation of the Kv6.4-mediated closed-state inactivation: suppression by 4-AP of the Kv2.1/Kv6.4 closed-state inactivation recovered a population of Kv2.1/Kv6.4 channels that was inactivated at resting conditions, i.e. at a holding potential of -80 mV. This modulation also resulted in a slower initiation and faster recovery from closed-state inactivation. Using chimeric substitutions between Kv6.4 and Kv9.3 subunits, we demonstrated that the lower half of the S6 domain (S6c) plays a crucial role in the 4-AP induced potentiation. These results demonstrate that KvS subunits modify the pharmacological response of Kv2 subunits when assembled in heterotetramers and illustrate the potential of KvS subunits to provide unique pharmacological properties to the heterotetramers, as is the case for 4-AP on Kv2.1/Kv6.4 channels.

Auxiliary KCNE subunits modulate both homotetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels.

The diversity of the voltage-gated K(+) (Kv) channel subfamily Kv2 is increased by interactions with auxiliary ß-subunits and by assembly with members of the modulatory so-called silent Kv subfamilies (Kv5-Kv6 and Kv8-Kv9). However, it has not yet been investigated whether these two types of modulating subunits can associate within and modify a single channel complex simultaneously. Here, we demonstrate that the transmembrane ß-subunit KCNE5 modifies the Kv2.1/Kv6.4 current extensively, whereas KCNE2 and KCNE4 only exert minor effects. Co-expression of KCNE5 with Kv2.1 and Kv6.4 did not alter the Kv2.1/Kv6.4 current density but modulated the biophysical properties significantly; KCNE5 accelerated the activation, slowed the deactivation and steepened the slope of the voltage-dependence of the Kv2.1/Kv6.4 inactivation by accelerating recovery of the closed-state inactivation. In contrast, KCNE5 reduced the current density ~2-fold without affecting the biophysical properties of Kv2.1 homotetramers. Co-localization of Kv2.1, Kv6.4 and KCNE5 was demonstrated with immunocytochemistry and formation of Kv2.1/Kv6.4/KCNE5 and Kv2.1/KCNE5 complexes was confirmed by Fluorescence Resonance Energy Transfer experiments performed in HEK293 cells. These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE5 subunits can be formed. In vivo, formation of such tripartite Kv2.1/Kv6.4/KCNE5 channel complexes might contribute to tissue-specific fine-tuning of excitability.

The subfamily-specific interaction between Kv2.1 and Kv6.4 subunits is determined by interactions between the N- and C-termini.

The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.1/Kv6.4 heterotetramers. The N-terminal T1 domain determines the subfamily-specific assembly of Kv1-4 subunits by preventing interactions between subunits that belong to different subfamilies. For Kv6.4, yeast-two-hybrid experiments showed an interaction of the Kv6.4 N-terminus with the Kv2.1 N-terminus, but unexpectedly also with the Kv3.1 N-terminus. We confirmed this interaction by Fluorescence Resonance Energy Transfer (FRET) and co-immunoprecipitation (co-IP) using N-terminal Kv3.1 and Kv6.4 fragments. However, full-length Kv3.1 and Kv6.4 subunits do not form heterotetramers at the plasma membrane. Therefore, additional interactions between the Kv6.4 and Kv2.1 subunits should be important in the Kv2.1/Kv6.4 subfamily-specificity. Using FRET and co-IP approaches with N- and C-terminal fragments we observed that the Kv6.4 C-terminus physically interacts with the Kv2.1 N-terminus but not with the Kv3.1 N-terminus. The N-terminal amino acid sequence CDD which is conserved between Kv2 and KvS subunits appeared to be a key determinant since charge reversals with arginine substitutions abolished the interaction between the N-terminus of Kv2.1 and the C-terminus of both Kv2.1 and Kv6.4. In addition, the Kv6.4(CKv3.1) chimera in which the C-terminus of Kv6.4 was replaced by the corresponding domain of Kv3.1, disrupted the assembly with Kv2.1. These results indicate that the subfamily-specific Kv2.1/Kv6.4 heterotetramerization is determined by interactions between Kv2.1 and Kv6.4 that involve both the N- and C-termini in which the conserved N-terminal CDD sequence plays a key role.

Cell type-specific spatial and functional coupling between mammalian brain Kv2.1 K+ channels and ryanodine receptors.

The Kv2.1 voltage-gated K+ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity-dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+ -release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy-immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR-mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca2+ /calcineurin-dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell- and circuit-specific mechanism for coupling intracellular Ca2+ release to phosphorylation-dependent regulation of Kv2.1 to dynamically impact intrinsic excitability.

The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels.

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.

Kv2.1 and silent Kv subunits underlie the delayed rectifier K+ current in cultured small mouse DRG neurons.

Silent voltage-gated K(+) (K(v)) subunits interact with K(v)2 subunits and primarily modulate the voltage dependence of inactivation of these heterotetrameric channels. Both K(v)2 and silent K(v) subunits are expressed in the mammalian nervous system, but little is known about their expression and function in sensory neurons. This study reports the presence of K(v)2.1, K(v)2.2, and silent subunit K(v)6.1, K(v)8.1, K(v)9.1, K(v)9.2, and K(v)9.3 mRNA in mouse dorsal root ganglia (DRG). Immunocytochemistry confirmed the protein expression of K(v)2.x and K(v)9.x subunits in cultured small DRG neurons. To investigate if K(v)2 and silent K(v) subunits are underlying the delayed rectifier K(+) current (I(K)) in these neurons, K(v)2-mediated currents were isolated by the extracellular application of rStromatoxin-1 (ScTx) or by the intracellular application of K(v)2 antibodies. Both ScTx- and anti-K(v)2.1-sensitive currents displayed two components in their voltage dependence of inactivation. Together, both components accounted for approximately two-thirds of I(K). A comparison with results obtained in heterologous expression systems suggests that one component reflects homotetrameric K(v)2.1 channels, whereas the other component represents heterotetrameric K(v)2.1/silent K(v) channels. These observations support a physiological role for silent K(v) subunits in small DRG neurons.