William B. Thornhill

The potassium channel subunit KV3.1b is localized to somatic and axonal membranes of specific populations of CNS neurons

Potassium channels play major roles in the regulation of many aspects of neuronal excitability. These channels are particularly well suited for such multiplicity of roles since there is a large diversity of channel types. This diversity contributes to the ability of specific neurons (and possibly different regions of the same neuron) to respond uniquely to a given input. Neuronal integration depends on the local response of spatially segregated inputs to the cell and the communication of these integration centers with the axon. Therefore, the functional implications of a given set of K+ channels varies depending on their precise location on the neuronal surface. Site- specific antibodies were utilized to characterize the distribution of KV3.1b, a subunit of voltage-gated K+ channels in CNS neurons. KV3.1b subunits are expressed in specific neuronal populations of the rat brain, such as cerebellar granule cells, projecting neurons of deep cerebellar nuclei, the substantia nigra pars-reticulata, the globus pallidus, and the ventral thalamus (reticular thalamic nucleus, ventral lateral geniculate and zona incerta). The KV3.1b protein is also present in various neuronal populations involved in the processing of auditory signals, including the inferior colliculus, the nuclei of the lateral lemniscus, the superior olive, and some parts of the cochlear nuclei; as well as in several other neuronal groups in the brainstem (e.g., in the oculomotor nucleus, the pontine nuclei, the reticulotegmental nucleus of the pons, trigeminal and vestibular nuclei, and the reticular formation) and subsets of neurons in the neocortex, the hippocampus and the caudate-putamen shown by double staining to correspond to neurons containing parvalbumin. KV3.1b subunits are localized predominantly in somatic and axonal membranes (particularly in axonal terminal fields) but are much less prominent in dendritic arborizations. This distribution is different than that of other subunits of voltage gated K+ channels and is consistent with a role in the modulation of action potentials. KV3.1b proteins have a cellular and subcellular distribution different than the related KV3.2 subunits which express in Xenopus oocytes currents similar to those expressed by KV3.1b.

Glycosylation Affects the Protein Stability and Cell Surface Expression of Kv1.4 but Not Kv1.1 Potassium Channels A PORE REGION DETERMINANT DICTATES THE EFFECT OF GLYCOSYLATION ON TRAFFICKING

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 (Watanabe, I., Wang, H. G., Sutachan, J. J., Zhu, J., Recio-Pinto, E. & Thornhill, W. B. (2003) J. Physiol. (Lond.) 550, 51–66) and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively (Zhu, J., Watanabe, I., Gomez, B. & Thornhill, W. B. (2001) J. Biol. Chem. 276, 39419–39427). Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1–S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.

Expression of Kv1.1 Delayed Rectifier Potassium Channels in Lec Mutant Chinese Hamster Ovary Cell Lines Reveals a Role for Sialidation in Channel Function

Kv1.1 potassium (K+) channels contain significant amounts of negatively charged sialic acids. To examine the role of sialidation in K+ channel function, Chinese hamster ovary cell lines deficient in glycosylation (Lec mutants) were transfected with rat brain Kv1.1 cDNA. The K+ channel was functionally expressed in all cell lines, but the voltage dependence of activation (V1/2) was shifted to more positive voltages and the activation kinetics were slower in the mutant cell lines compared with control. A similar positive shift in V1/2 was recorded in control cells expressing Kv1.1 following treatment with sialidase or by raising extracellular Ca2+. In contrast, these treatments had little or no effect on the Lec mutants, which indicates that channel sialic acids appear to be the negative surface charges sensitive to Ca2+. The data suggest that sialic acid addition modifies Kv1.1 channel function, possibly by influencing the local electric field detected by its voltage sensor, but that these carbohydrates are not required for cell surface expression.

Glycosylation affects rat Kv1.1 potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism

The effect of glycosylation on Kv1.1 potassium channel function was investigated in mammalian cells stably transfected with Kv1.1 or Kv1.1N207Q. Macroscopic current analysis showed that both channels were expressed but Kv1.1N207Q, which was not glycosylated, displayed functional differences compared with wild‐type, including slowed activation kinetics, a positively shifted V1/2, a shallower slope for the conductance versus voltage relationship, slowed C‐type inactivation kinetics, and a reduced extent of and recovery from C‐type inactivation. Kv1.1N207Q activation properties were also less sensitive to divalent cations compared with those of Kv1.1. These effects were largely due to the lack of trans‐Golgi added sugars, such as galactose and sialic acid, to the N207 carbohydrate tree. No apparent change in ionic current deactivation kinetics was detected in Kv1.1N207Q compared with wild‐type. Our data, coupled with modelling, suggested that removal of the N207 carbohydrate tree had two major effects. The first effect slowed the concerted channel transition from the last closed state to the open state without changing the voltage dependence of its kinetics. This effect contributed to the G‐V curve depolarization shift and together with the lower sensitivity to divalent cations suggested that the carbohydrate tree and its negatively charged sialic acids affected the negative surface charge density on the channels extracellular face that was sensed by the activation gating machinery. The second effect reduced a cooperativity factor that slowed the transition from the open state to the closed state without changing its voltage dependence. This effect accounted for the shallower G‐V slope, and contributed to the depolarized G‐V shift, and together with the inactivation changes it suggested that the carbohydrate tree also affected channel conformations. Thus N‐glycosylation, and particularly terminal sialylation, affected Kv1.1 gating properties both by altering the surface potential sensed by the channels activation gating machinery and by modifying conformational changes regulating cooperative subunit interactions during activation and inactivation. Differences in glycosylation pattern among closely related channels may contribute to their functional differences and affect their physiological roles.

Phosphorylation of a K+ channel alpha subunit modulates the inactivation conferred by a beta subunit. Involvement of cytoskeleton.

Voltage-gated K+ channels isolated from mammalian brain are composed of α and β subunits. Interaction between coexpressed Kv1.1 (α) and Kvβ1.1 (β) subunits confers rapid inactivation on the delayed rectifier-type current that is observed when α subunits are expressed alone. Integrating electrophysiological and biochemical analyses, we show that the inactivation of the αβ current is not complete even when α is saturated with β, and the αβ current has an inherent sustained component, indistinguishable from a pure α current. We further show that basal and protein kinase A-induced phosphorylations at Ser-446 of the α protein increase the extent, but not the rate, of inactivation of the αβ channel, without affecting the association between α and β. In addition, the extent of inactivation is increased by agents that lead to microfilament depolymerization. The effects of phosphorylation and of microfilament depolymerization are not additive. Taken together, we suggest that phosphorylation, via a mechanism that involves the interaction of the αβ channel with microfilaments, enhances the extent of inactivation of the channel. Furthermore, phosphorylation at Ser-446 also increases current amplitudes of the αβ channel as was shown before for the α channel. Thus, phosphorylation enhances in concert inactivation and current amplitudes, thereby leading to a substantial increase in A-type activity.

The glycosylation state of Kv1.2 potassium channels affects trafficking, gating, and simulated action potentials.

We presented evidence previously that decreasing the glycosylation state of the Kv1.1 potassium channel modified its gating by a combined surface potential and a cooperative subunit interaction mechanism and these effects modified simulated action potentials. Here we continued to test the hypothesis that glycosylation affects channel function in a predictable fashion by increasing and decreasing the glycosylation state of Kv1.2 channels. Compared with Kv1.2, increasing the glycosylation state shifted the V(1/2) negatively with a steeper G-V slope, increased activation kinetics with little change in deactivation kinetics or in their voltage-dependence, and decreased the apparent level of C-type inactivation. Decreasing the glycosylation state had essentially the opposite effects and shifted the V(1/2) positively with a shallower G-V slope, decreased activation kinetics (and voltage-dependence), decreased deactivation kinetics, and increased the apparent level of C-type inactivation. Single channel conductance was not affected by the different glycosylation states of Kv1.2 tested here. Hyperpolarized or depolarized shifts in V(1/2) from wild type were apparently due to an increased or decreased level of channel sialylation, respectively. Data and modeling suggested that the changes in activation properties were mostly predictable within and between channels and were consistent with a surface potential mechanism, but those on deactivation properties were not predictable and were more consistent with a conformational mechanism. Moreover the effect on the deactivation process appeared to be channel-type dependent as well as glycosylation-site dependent. The glycosylation state of Kv1.2 also affected action potentials in simulations. In addition, preventing N-glycosylation decreased cell surface Kv1.2 expression levels by approximately 40% primarily by increasing partial endoplasmic reticulum retention and this effect was completely rescued by Kv1.4 subunits, which are glycosylated, but not by cytoplasmic Kvbeta2.1 subunits. The nonglycosylated Kv1.2 protein had a similar protein half-life as the glycosylated protein and appeared to be folded properly. Thus altering the native Kv1.2 glycosylation state affected its trafficking, gating, and simulated action potentials. Differential glycosylation of ion channels could be used by excitable cells to modify cell signaling.

Determinants Involved in Kv1 Potassium Channel Folding in the Endoplasmic Reticulum, Glycosylation in the Golgi, and Cell Surface Expression

Kv1.1 and Kv1.4 potassium channels are expressed as mature glycosylated proteins in brain, whereas they exhibited striking differences in degree of trans-Golgi glycosylation conversion and high cell surface expression when they were transiently expressed as homomers in cell lines. Kv1.4 exhibited a 70%trans-Golgi glycosylation conversion, whereas Kv1.1 showed none, and Kv1.4 exhibited a ∼20-fold higher cell surface expression level as compared with Kv1.1. Chimeras between Kv1.4 and Kv1.1 and site-directed mutants were constructed to identify amino acid determinants that affected these processes. Truncating the cytoplasmic C terminus of Kv1.4 inhibited its trans-Golgi glycosylation and high cell surface expression (as shown by Li, D., Takimoto, K., and Levitan, E. S. (2000) J. Biol. Chem. 275, 11597–11602), whereas truncating this region on Kv1.1 did not affect either of these events, indicating that its C terminus is not a negative determinant for these processes. Exchanging the C terminus between these channels showed that there are other regions of the protein that exert a positive or negative effect on these processes. Chimeric constructs between Kv1.4 and Kv1.1 identified their outer pore regions as major positive and negative determinants, respectively, for both trans-Golgi glycosylation and cell surface expression. Site-directed mutagenesis identified a number of amino acids in the pore region that are involved in these processes. These data suggest that there are multiple positive and negative determinants on both Kv1.4 and Kv1.1 that affect channel folding,trans-Golgi glycosylation conversion, and cell surface expression.

Inactivation of a Voltagedependent K+ Channel by β Subunit MODULATION BY A PHOSPHORYLATION-DEPENDENT INTERACTION BETWEEN THE DISTAL C TERMINUS OF α SUBUNIT AND CYTOSKELETON

Kv1.1/Kvβ1.1 (αβ) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the α subunit at Ser-446, which impairs the interaction of the channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of α, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C terminus of α. Truncation of the last six amino acids resulted in αβ channels with an extent of inactivation up to 2.5-fold larger and its further enhancement by cytochalasins being reduced 2-fold. The wild-type channels exhibited strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the wild-type channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of proteins, we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phosphorylation of the channel, it regulates the extent of inactivation conferred by the β subunit.

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvβ1.1 subunits modulated by G protein βγ subunits

Modulation of A‐type voltage‐gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed‐rectifier Kv1.1 channel can be converted to A‐type upon association with Kvβ1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gβ1γ2 subunits concomitantly with the channel (composed of Kv1.1 and Kvβ1.1 subunits), but not after the channels expression in the plasma membrane, increases the extent of conversion to A‐type. Conversely, scavenging endogenous Gβγ by co‐expression of the C‐terminal fragment of the β‐adrenergic receptor kinase reduces the extent of conversion to A‐type. The effect of Gβγ co‐expression is occluded by treatment with dihydrocytochalasin B, a microfilament‐disrupting agent shown previously by us to enhance the extent of conversion to A‐type, and by overexpression of Kvβ1.1. Gβ1γ2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvβ1.1. Co‐expression of Gβ1γ2 causes co‐immunoprecipitation with Kv1.1 of more Kvβ1.1 subunits. Thus, we suggest that Gβ1γ2 directly affects the interaction between Kv1.1 and Kvβ1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A‐type conversion.

Allowed N-glycosylation sites on the Kv1.2 potassium channel S1-S2 linker: implications for linker secondary structure and the glycosylation effect on channel function.

N-glycosylation is a post-translational modification that plays a role in the trafficking and/or function of some membrane proteins. We have shown previously that N-glycosylation affected the function of some Kv1 voltage-gated potassium (K+) channels [Watanabe, Wang, Sutachan, Zhu, Recio-Pinto and Thornhill (2003) J. Physiol. (Cambridge, U.K.) 550, 51-66]. Kv1 channel S1-S2 linkers vary in length but their N-glycosylation sites are at similar relative positions from the S1 or S2 membrane domains. In the present study, by a scanning mutagenesis approach, we determined the allowed N-glycosylation sites on the Kv1.2 S1-S2 linker, which has 39 amino acids, by engineering N-glycosylation sites and assaying for glycosylation, using their sensitivity to glycosidases. The middle section of the linker (54% of linker) was glycosylated at every position, whereas both end sections (46% of linker) near the S1 or S2 membrane domains were not. These findings suggested that the middle section of the S1-S2 linker was accessible to the endoplasmic reticulum glycotransferase at every position and was in the extracellular aqueous phase, and presumably in a flexible conformation. We speculate that the S1-S2 linker is mostly a coiled-loop structure and that the strict relative position of native glycosylation sites on these linkers may be involved in the mechanism underlying the functional effects of glycosylation on some Kv1 K+ channels. The S3-S4 linker, with 16 amino acids and no N-glycosylation site, was not glycosylated when an N-glycosylation site was added. However, an extended linker, with an added N-linked site, was glycosylated, which suggested that the native linker was not glycosylated due to its short length. Thus other ion channels or membrane proteins may also have a high glycosylation potential on a linker but yet have similarly positioned native N-glycosylation sites among isoforms. This may imply that the native position of the N-glycosylation site may be important if the carbohydrate tree plays a role in the folding, stability, trafficking and/or function of the protein.