BACKGROUND
voltage-dependent potassium  channels are regulators of neuronal excitability.
the channels are responsible for maintaining the resting potential of cells, they determine the width and maximum amplitude of the action potential, and they govern the interpulse interval or timing patterns of firing in order to relay sensory information to the brain or coordinate motor output  <cit> .
as recently reviewed by kaczmarek  <cit> , kv channels may lead a "double life" by possessing non-conducting functions, which allow them to participate in coupled biochemical reactions or communicate directly with cytoplasmic and nuclear signaling pathways.
kv <dig> , a member of the shaker subfamily of kv channels, is particularly well poised to participate in multiple cell signaling pathways given a number of molecular motifs that serve as protein-protein interaction domains in the n and c terminal aspects of the channel protein .
multiple and different combinations of tyrosine residues become phosphorylated upon activation of cellular and receptor tyrosine kinase signaling cascades to elicit changes in ion channel current magnitude, inactivation kinetics, or cumulative/use-dependent inactivation  <cit> .
further levels of complexity exist, however, because the channel is not just a substrate for tyrosine phosphorylation.
the phosphorylated tyrosine residue now become recognition motifs for a variety of src homology  <dig>  domain containing proteins  <cit>  that are down-stream signaling molecules, or adaptor proteins, without catalytic activity  <cit> , and which functionally subserve to "modulate the modulation"  <cit> .
two proline-rich domains have been shown to serve as phosphorylation-independent binding domains for src homology  <dig>  containing proteins and ubiquitin ligases to change kv <dig>  channel clustering and surface expression  <cit> .
gene-targeted deletion of kv <dig>  demonstrates that the channel's associated scaffold of kinases and adaptor proteins becomes unbalanced in the absence of the channel gene, whereby there is a significant increase in the expression of these modulatory cell signaling proteins, including neurotrophin receptor tyrosine kinase b , the cellular kinase src, the adaptor proteins 14-3- <dig>  neuronal src homology and collagen , postsynaptic density protein- <dig> , and the growth factor receptor-bound protein  <dig>   <cit> .
because kv <dig>  protein has an abundance of molecular modules for protein-protein interactions , it is not surprising to discover a diversity of non-conducting functions following targeted deletion of the core of the scaffold   <cit> .
this study focuses upon two adaptor proteins enriched in the olfactory bulb, namely grb <dig> and nshc.
the adaptor protein grb <dig> is a member of a superfamily of adaptor proteins that also includes grb <dig>  grb <dig>  and mig- <dig>  <cit> .
the characterization of conserved molecular architecture of this family has shown that members all contain a c-terminal sh <dig> domain, a central segment containing a pleckstrin homology domain , and a principle binding region for association with insulin-like growth factor receptor type  <dig> that also acts to stabilize insulin receptor kinase  binding to the sh <dig> domain .
the n terminal domain of grb <dig> is less well conserved but contains multiple pxxp domains that serve as putative binding sites for sh <dig> domain containing proteins  <cit>  .
while grb <dig> was originally discovered to be an interacting protein with the epidermal growth factor receptor   <cit>  and later found to be a negative regulator of the ir via ubiquitination  <cit> , it may also interact with other rtks.
grb <dig> has been linked with human growth retardation and type  <dig> diabetes when upregulated or mutated  <cit> , which alters grb <dig> protein-protein interactions.
the adaptor protein nshc belongs to a family that includes three major genes, shca , shcb , and shcc .
all shc isoforms possess two distinct domains that bind phosphotyrosine containing sequences, namely, the phosphotyrosine binding domain  and the sh <dig> domain, and a central domain  that contains multiple tyrosine phosphorylation sites  <cit>  .
nshc is specifically enriched in adult brain and has been found to direct survival and phenotypic maturation of neurons via association with neurotrophin receptors  <cit> .
more recently, nshc has been also implicated in the modulation of hippocampal synaptic plasticity via modulation of the nmda receptor, independent of neurotrophin receptor signaling  <cit> .
previous site-directed mutagenesis studies in heterologous expression systems, have demonstrated that bdnf-activation of trkb kinase phosphorylates kv <dig>  at three tyrosine  residues or clusters in the n- and c-termini of the channel, y111– <dig>  y <dig>  y <dig>  to cause suppression of current magnitude without changes in channel inactivation or deactivation kinetics  <cit> .
our current strategy was to co-express channel and kinase with one of two adaptor proteins that varied in their reported interactions:  an adaptor protein that was not known to directly associate with the neurotrophin receptor or the channel , and  an adaptor protein that was known to bind to y <dig>  of the neurotrophin receptor .
unexpectedly, we discovered that nshc adaptor proteins reportedly characterized to interact with rtks also used kv <dig>  channel for a binding partner directly.
grb <dig> did not interact with either the channel or kinase, but could indirectly modulate the channel by changing its surface expression.
in native ob, however, both adaptor proteins co-ip with kv <dig>  and trkb kinase.
our data suggest that there is a complex regulation between the channel, the kinase, and adaptor proteins such that the expression of kv <dig>  down regulates that of trkb kinase, while the expression of grb <dig> likely acts to induce ubiquitination of the channel thus decreasing channel surface expression.
RESULTS
co-localization of adaptor proteins and kv <dig>  channel in the olfactory bulb
although the goal of our study was to define the molecular targets for interaction of kv <dig>  plus trkb kinase in the presence of adaptor proteins using a well-defined heterologous expression system , it was important to ascertain the physiological significance of such interactions by first exploring the potential co-localization of the adaptor proteins with the channel in native brain.
kv <dig>  has a limited distribution in the central nervous system, where it has been studied primarily in regions of highest expression, namely in the olfactory bulb , the olfactory cortex, and the dentate gyrus of the hippocampus  <cit> .
we have previously reported the expression of several adaptor proteins  in cytoplasmic lysates of the ob by western blot analysis  <cit>  as well as the co-localization and co-ip of the channel and trkb kinase in the ob and olfactory cortex  <cit> .
thus, we explored co-localization of kv <dig>  with that of nshc  and grb <dig>  in the ob of postnatal day 20– <dig> mice where kv <dig>  channel properties are well characterized  <cit>  and it is known that modulation of kv <dig>  alters olfactory threshold  <cit> .
kv <dig>  and nshc demonstrated greatest overlap of expression in the peripheral structures surrounding the glomeruli  and weaker co-localization patterns in the external plexiform layer , the mitral cell layer , the internal plexiform layer , and the granule cell layer .
kv <dig>  and grb <dig> demonstrated greatest overlap of expression in the ipl and gcl  and weaker co-localization patterns in some fibers surrounding the glomeruli .
adaptors can directly co-ip with kv <dig>  channel independent of trkb kinase
since both adaptors demonstrated a degree of colocalization with kv <dig>  in native brain tissue, we sought more rigorous conformation for a potential protein-protein interaction using hek <dig> cells in which site specific mutation of the channel could be performed to map the molecular targets for channel-adaptor scaffolding.
we have previously demonstrated that bdnf-induced kv <dig>  current suppression is mediated through tyrosine phosphorylation of three sites in the n and c termini of the channel, y111– <dig>  y <dig> and y <dig>  <cit> .
since both nshc and grb <dig> have sh <dig> domains that could target any or a combination of these tyrosine residues upon bdnf-induced trkb phosphorylation, we wanted to test whether either adaptor protein could associate with the channel under bdnf-stimulated conditions.
as demonstrated in fig.
4a , immunoprecipitation of myc-tagged kv <dig>  , followed by western analysis and blotting for shc  labels predominantly a single  <dig> kda band in hek  <dig> cells that were only co-transfected with myckv <dig>  + shc .
therefore, the channel and the shc adaptor protein form a protein-protein interaction complex independent of bdnf-induced phosphorylation of the channel or at least in the absence of trkb kinase.
inclusion of trkb kinase or y490f trkb  cdna into the transfection scheme does not prevent or increase the channel/adaptor interaction , further exemplifying the lack of requirement of trkb in the channel/adaptor complex.
as shown in fig.
4b, we were not able to co-immunoprecipitate trkb and grb <dig>  or kv <dig>  and grb <dig> , indicating that unlike nshc adaptor protein  <cit> , grb <dig> does not  associate with the kinase.
also, note the expression of native levels of adaptor proteins, apparently endogenous to hek  <dig> cells in lanes untransfected with shc or grb <dig> cdna .
adaptor/kinase/channel scaffold can be precipitated from olfactory bulb and hippocampus
given our ability to co-immunoprecipitate nshc and kv <dig>  channel, we next explored whether the proteins interacted in native tissue by using two regions known to express kv <dig>   <cit> .
as demonstrated in fig.
<dig>  immunoprecipitation of kv <dig> , followed by western analysis and blotting for anti-trkb, anti-shc, or anti-grb <dig>  labels protein bands of predicted molecular mass for the kinase, and adaptors respectively.
as we have previously reported for the olfactory bulb  <cit> , trkb labeling demonstrated a greater intensity for the full-length receptor protein  over that of its truncated form  that lacks a kinase domain .
the predicted molecular mass for shc is multiple depending upon the variant  and in previous cytosolic fractions we have observed all three variants in olfactory bulb preparations  <cit> .
it is interesting that in the hek  <dig> cells, the molecular migration was closest to the  <dig> kda variant, whereas in the olfactory bulb, the variant associated with kv <dig>   was different than those associated with trkb  .
moving to the hippocampus, the variants  forming a protein-protein interaction with kv <dig>  were also different than that in the olfactory bulb .
it is also noteworthy, that while grb <dig> could not be consistently co-immunoprecipitated with either the kinase nor the channel via heterologous expression , both were complexed with the adaptor protein in the olfactory bulb and the hippocampus .
it is possible in the hek  <dig> cells that the interaction is at the limit of our detection.
it may also suggest either a a missing scaffolding component in the hek  <dig> cells that is essential for protein-protein interactions with the grb <dig> adaptor or a different regulation of grb <dig> in neurons.
grb <dig> decreases surface expression density of the channel
we have previously reported that kv <dig>  channel modulation can be perturbed by adaptor proteins via disruption of src- or insulin-induced kv <dig>  phosphorylation  <cit> .
in the cellular tyrosine kinase signaling pathway , adaptor proteins that do not disrupt tyrosine phosphorylation have no effect on channel function; however, those that decrease tyrosine phosphorylation can relieve kinase-induced current suppression.
in the receptor tyrosine kinase signaling pathway , adaptor proteins that disrupt tyrosine phosphorylation relieve both current suppression and simultaneously act to increase the rate of channel inactivation.
for the neurotrophin pathway , we questioned how the degree of channel phosphorylation as modified by an adaptor protein might be linked to function.
thus, we immunoprecipitated kv <dig>  channel plus trkb kinase under various transfection conditions that included either shc or grb <dig> adaptors .
interestingly, inclusion of nshc in the transfection scheme  increased the y phosphorylation of the channel over that observed under the co-transfection condition alone  .
moreover, elimination of the y <dig> trkb site via substitution with trkbshc- in the channel/kinase/adaptor transfection scheme  completely prevented tyrosine phosphorylation of the channel.
basal phosphorylation of kv <dig>   was higher than expected or reported in our previous studies  <cit> , but may be attributed to the solution used as a vehicle carrier  that may have slightly depolarized the cells .
none the less, all basal phosphorylation could be completely eliminated in the absence of trkb  or via substitution with a dead trkb kinase construct in which the catalytic domain was truncated   <cit> .
most interestingly, inclusion of grb <dig> in the transfection scheme  not only completely eliminated the y phosphorylation of the channel over that observed in the co-transfection condition , blotting the lysates with anti-kv <dig>  to control for expression of the channel, revealed that grb <dig> was greatly reducing the expression of kv <dig>  .
the degree of bdnf-induced kv <dig>  phosphorylation in each transfection condition was quantified by densitometry, normalized to that of the kv <dig>  channel transfection alone, and statistically compared in the bar plot of fig.
6c .
expression levels of grb <dig>  kv <dig> , and trkb are cross-regulated
to further explore the robustness of our finding of grb <dig> downregulation of kv <dig> , we compared the surface fraction of kv <dig>  channels  and the total kv <dig>  pool  in the presence and absence of grb <dig>  as shown in fig.
7a, b both the surface fraction of kv <dig>  channels as well as the total pool of kv <dig>  is dramatically down regulated by grb <dig> adaptor protein .
grb <dig> also had the capacity to down regulate kv <dig>  protein expression in the presence of trkb kinase .
in conjunction with the ability to downregulate total channel protein, immunocytochemical experiments demonstrated a significant reduction in fluorescent signal at the membrane under grb <dig> co-transfection conditions .
albeit lack of grb10/kv <dig>  co-immunoprecipitation in vitro, if grb <dig> was downregulating the expression of kv <dig>  channel via the sh <dig> recognition domains on kv <dig> , then substitution of y to f mutations in the n- and c-terminal aspects of the channel should completely eliminate any reduction in expression.
single point mutagenesis at the selected y residues of this study does not alter expression of the derived channel mutants as determined by either western analysis or electrophysiology  <cit> .
western analysis of hek  <dig> cells in which grb <dig> was co-transfected with wild-type or various y to f channel mutants revealed that residues yyy111– <dig> and y <dig> represent important residues for regulation between channel and adaptor protein .
removal of these residues prevented kv <dig>  downregulation by grb <dig> co-transfection.
mutation of residues y <dig> and y <dig> retained the trend for down-regulation of kv <dig> , thus these residues are likely not involved in the adaptor's regulation of channel expression.
next, we tested whether channel conductance was necessary for grb <dig> down-regulation of kv <dig>  by substituting the pore-conducting mutant w386f kv <dig>   <cit> .
as shown in fig.
7d and 7e, mutation of the pore had no effect in the adaptor's ability to down-regulate kv <dig>  channel expression.
finally, we tested whether the grb <dig> down-regulation of kv <dig>  was specific to this particular shaker subfamily member or could be extended to other potassium channels.
substitution in the transfection scheme of either kv <dig>  or kv <dig> , two family members that are also expressed in olfactory bulb neurons  <cit> , elicited no change in channel expression when co-transfected with grb <dig> .
furthermore, expression of general structural proteins  also demonstrated no change in expression in conditions of grb <dig> transfection.
adaptor proteins grb <dig> and nshc, respectively decreased and increased the expression of kv <dig>  channel.
of noteworthy regulation, curiously it was observed that co-transfection with kv <dig>  channel protein decreased both the expression of trkb  as well as the phosphorylated state of the kinase when stimulated with bdnf .
this was not only true for trkb, but also was observed for a different, receptor tyrosine kinase .
expression of kv <dig>  channel also decreased the expression of ir kinase protein and its concomitant phosphorylation by insulin .
the degree of kv <dig>  expression in the presence and absence of trkb or ir kinase was quantified by densitometry, normalized to that of the kv <dig>  channel transfection alone, and statistically compared in the bar plot of fig.
8b .
these data pose a complex balance between channel, kinase, and adaptor whereby the level of an adaptor protein could down or upregulate expression of the channel, which could in turn, dial in the degree of phosphorylation of the kinase .
bdnf-stimulated channel modulation is blocked by grb <dig> and nshc adaptor proteins
if the two adaptor proteins differentially co-immunoprecipitated with kv <dig>  channel, one might predict a functional difference in their ability to modulate the channel as determined through standard patch-clamp electrophysiology.
hek <dig> cells were thus transiently transfected with kv <dig>  plus trkb kinase in the presence or absence of nshc or grb <dig> adaptor protein and monitored for modulation of biophysical properties during a twenty minute acute stimulation with brain-derived neurotrophic factor .
approximately  <dig> hours post-transfection, macroscopic outward currents were recorded in the cell-attached configuration.
cells were voltage-clamped at - <dig> mv and stepped to a depolarizing potential of + <dig> mv for  <dig> second to activate kv <dig> .
table  <dig> reports kv <dig>  peak current amplitude, inactivation time constant , deactivation time constant , voltage at half-activation , and voltage dependence  for a population of recorded patches under various transfection conditions pre  and post  growth factor application.
twenty minute application of bdnf had no effect on patches transfected with kv <dig>  channel alone .
this control confirms that application of ligand alone in the absence of the receptor  fails to modulate kv <dig> .
inclusion of kv <dig>  plus trkb significantly increased peak current amplitude without altering kinetic properties .
peak current amplitude was  <dig> +/-  <dig> pa  in the kv <dig> -only control transfection condition and  <dig> +/-  <dig> pa  in the kv <dig>  plus trkb kinase condition, confirming a statistically significant 90% increase in kv <dig>  current in the presence of the kinase .
we have previously reported that trkb kinase co-expression in the absence of bdnf stimulation increases the channel half-life at the plasma membrane  <cit>  so the increase in kv <dig>  current was as expected.
secondarily, these records demonstrate typical stability of the patch over the recording interval for the co-transfection condition .
inclusion of  <dig> ng/μl bdnf in the patch electrode  evoked a significant suppression of peak current amplitude of kv <dig>  to 85% of its original current  that was relieved by co-transfection of either adaptor protein .
in addition, we noted that initial peak current amplitude  of kv <dig>  in the co-transfection condition ) was significantly less in the presence of nshc ) but was unchanged in the presence of grb <dig> ) .
regardless of the pre-stimulation peak current, inclusion of either adaptor protein, prevented bdnf-induced kv <dig>  current suppression.
fig.
9e is a histogram representation of the normalized  bdnf-induced kv <dig>  current suppression ) that is relieved in the presence of shc or grb <dig> .
for grb <dig>  the relief in bdnf-induced current suppression appeared to be coincident with a slight shift in kv <dig>  voltage dependence.
there was a  <dig> to  <dig> mv left shift in the voltage at half-maximum activation  in the presence of trkb plus grb <dig>  as calculated from a boltzman fit of the tail currents when cells were held at - <dig> mv and stepped to + <dig> mv in increments of  <dig> mv using a pulse duration of  <dig> ms .
because this was not a robust effect and was somewhat variable even in the absence of an adaptor protein, this biophysical property was not further studied.
hek  <dig> cells were transfected with cdnas encoding kv <dig>  ± trkb ki nase ± adaptor protein.
several voltage-stimulating paradigms were completed  to collect the tabled biophysical properties by cell-attached patch-clamp recording.
mean values  for peak current amplitude , inactivation time constant , deactivation time constant , voltage at half activation , and slope  of the voltage dependence were compared across various transfection conditions at the start of the patch recording  versus twenty minutes following  <dig> ng/μg bdnf  using a paired t-test, α ≤  <dig> .
* = significantly different.
nshc signaling at y <dig> trkb is target for blocked modulation of kv <dig>  channel
to our knowledge there no reported direct interactions between trkb and grb <dig>  so we focused our study upon disruption of known targets of shc binding to trkb kinase.
the sh <dig> domain  of nshc is known to bind phosphorylated y <dig> trkb at the npqpy recognition motif  <cit> .
if the molecular target for nshc perturbation of bdnf-induced kv <dig>  current suppression was soley y <dig> on trkb kinase, then destruction of this binding site  should regain bdnf modulation of kv <dig>  .
what we found by substituting the trkb kinase mutant construct, however, was an intermediate effect.
co-transfection of kv <dig>  + trkbshc- plus nshc cdna exhibited bdnf-induced current suppression of kv <dig>   versus  <dig> +/-  <dig> pa  that did not reach statistical significance  compared with that observed with kv <dig>  + trkb co-expression  versus  <dig> +/-  <dig> pa ; significantly different paired t-test, α ≤  <dig> ; n=  <dig>  84% suppression) .
if, however, we substituted grb <dig> in the transfection scheme instead of nshc , we observed retention of bdnf-induced current suppression that was not relieved by the adaptor protein  versus  <dig> +/-  <dig> pa ; significantly different paired t-test, α ≤  <dig> ; n =  <dig>  71% suppression) .
although grb <dig> has never been reported to directly interact with trkb kinase signaling cascades, and we also do not find that this adaptor co-immunoprecipitates with the kinase in vitro , residue y <dig> of trkb kinase is an important target for nshc, and its mutation appears to somehow uncouple grb <dig> modulation of the channel.
with the removal of y <dig> of trkb kinase, grb <dig> can no longer relieve bdnf-induced current suppression of the channel .
interestingly, although neither adaptor protein induces a change in peak current amplitude of kv <dig>  alone , grb <dig> also evoked a faster rate of channel inactivation  versus  <dig> +/-  <dig> msec  as demonstrated in fig.
10e and statistically compared in table  <dig>
hek  <dig> cels were transfected with cdnas encoding kv <dig>  +trkb kinase or trkbshc-  ± adaptor protein.
several voltage-stimulating paradigms were completed  to collect the tabled biophysical properties by cell-attached patch-clamp recording.
bdnf stimulation, notations, and statistical analysis as in table  <dig>
discussion
kv <dig>  is a substrate for multiple tyrosine kinase signaling cascades including ir, trkb, and src kinase  <cit> .
each exerts the phosphorylation of multiple tyrosine residues, and different combinations of residues, to produce a unified response, of kv <dig>  current suppression.
inclusion of adaptor proteins causes perturbation of the channel current suppression  <cit> , and in the case of neurotrophin signaling, we find that disruption of the channel modulation is not directly correlated to the extent of channel phosphorylation.
the scenario is more complex.
this may be attributed to the fact that the interaction between kinase and channel is no longer a binary relationship, with the added adaptor having the capacity to bind to both channel and kinase to elicit different responses.
not only can an adaptor protein alter how the channel will respond to neurotrophin-induced current suppression, the adaptors alter expression levels of the channel and the channel itself can downregulate trkb expression and its phosphorylated state.
such a dynamic equilibrium between kv <dig> /trkb/adaptors may allow a full spectrum of modulation of channel properties dependent upon the compliment of proteins co-expressed in a neuron at a given physiological state.
biochemically, the degree of channel phosphorylation by bdnf-induced trkb activation is no longer directly related to the degree of functional current suppression when adaptors are added to the mix.
this is very different than what is reported for the cellular tyrosine kinase, src.
here, phosphorylation was directly linked to the degree of current suppression and one could predict the modulation of the selected adaptor protein based upon how it influenced tyrosine phosphorylation of the channel  <cit> .
this is in stark contrast to rtks.
the addition of nshc to kv <dig>  plus trkb transfected conditions induced an increase in both channel expression as well as bdnf-induced channel phosphorylation whereas the addition of grb <dig> resulted in the opposite.
despite the fact that the adaptors evoke an opposite response biochemically, the functional ramification is identical, the prevention of bdnf-induced current suppression.
in fact, nshc/grb have structurally similar sh <dig> domains whereas psd- <dig> lacks such phosphorylation-dependent interactive domains  <cit> , and yet each has the capacity to functionally perturb the bdnf-induced current suppression.
this might suggest that direct interaction of the adaptor and the channel may play a predominant role in preventing kinase modulation and that changed phosphorylation is a secondary effect.
in the case of the two sh <dig> domain containing adaptor proteins, nshc and grb <dig>  binding of y <dig> trkb by nshc is an additional key regulatory site impacting modulation of the channel.
certainly there are endogenous levels of both nshc and grb <dig> expressed in hek  <dig> cells, thus our kv <dig>  + trkb conditions are not completed devoid of adaptor protein, however, when nshc is co-transfected with these conditions, binding of y <dig> trkb induces a block of bdnf-induced channel suppression.
kv <dig>  channel expression is changed in the presence of trkb kinase without bdnf stimulation and now we can also see that grb <dig> adaptor significantly modifies surface expression of kv <dig> .
with trkb kinase co-expression, there is a correlate increase in channel surface expression, increase in channel protein expression, and increase in channel macroscopic current  <cit> .
interestingly, with grb <dig> co-expression  there is oppositely a decrease in channel surface expression, a decrease in protein expression of the channel, but no change in channel macroscopic current, rather an increased rate of channel inactivation.
the adaptor/channel do not form a complex in vitro that we can detect, nevertheless, co-expression apparently changes surface distribution and channel density but not current magnitude or conduction.
this would imply that there must be many kv <dig>  channels that are not contributing to current.
such a pool of silent or "sleeping channels" has been reported for specific kv subfamily members   <cit>  and uncovered for other potassium channels during frac   <cit> .
our data  <cit>  and that reported by sun et al.
<cit>  support that molecular/protein density of a channel may not always exhibit linearity with functional density.
based upon the crystal structure of grb <dig> sh <dig> domain determined at  <dig>  Å resolution, it is suggested that binding of grb <dig> is dimeric, using turn containing phosphotyrosine sequences  <cit> .
it is unknown  if the kv <dig>  channel contains turn containing phosphotyrosine motifs unless the tetrameric structure of the subunits permitted such an alignment, which would be contrary to positioning of the α transmembrane helices  <dig> and  <dig> for kv <dig> , a shaker family member for which crystal structure has been reported  <cit> .
on the basis of structural analysis and previous biochemical studies for the adaptor, grb <dig> and grb <dig> have a partially impaired ability to bind phosphotyrosine-containing ligands due to a non-glycyl residue at the end of the bd loop and lack of a p +  <dig> binding pocket  <cit> .
these combined data are consistent with our findings that we cannot detect grb <dig> and kv <dig>  co-immunoprecipition in vitro, albeit the fact that channel/adaptor functional interactions appear to modulate channel expression and can be found as a complex in unstimulated native tissues.
the fact that mutation of several y residues on the n and c terminal aspects of the channel clearly perturbs the negative modulatory affect of grb <dig> on channel expression, indicates a specific modulatory role that does not require tight association.
moreover, the modulation does not require channel activity, or at least functional ion conductance.
while all y to f channel mutations blocked grb <dig> down regulation of kv <dig>  expression, yyy111-113fff kv <dig>  and y449f kv <dig>  appear to be the most robust at blocking down regulation.
both of these sites  were previously reported as targets for bdnf-induced kv <dig>  current suppression and phosphorylation  <cit>  but only y <dig> has a large hydrophobic m downstream of the phosphotyrosine, reported as a preferred grb motif  <cit> .
it has previously been reported that the e <dig> ubiquitin protein ligase called nedd4- <dig> regulates voltage-gated ion channels, including nav, kir, and kv family members  <cit>  via targeting phosphotyrosine motifs located in the c-terminal aspect of the channel.
it has also been demonstrated that the sh <dig> domains of grb <dig> form a constitutive complex with nedd4- <dig> so as to mediate ligand-dependent ubiquitination of igf-ir  <cit> .
since co-expression of nedd4- <dig> with kv <dig>  in xenopus oocytes induces a strong suppression of kv <dig>  current  <cit>  we conjecture that grb <dig> might bind to endogenous nedd4- <dig> in our system to cause ubiquitination of kv <dig>  and/or trkb kinase.
this might explain a potential mechanism for decreased kv <dig>  surface expression in the presence of grb <dig> and correlate decreased channel tyrosine phosphorylation.
we have shown that trkb and grb <dig> act as modulators of channel expression, while others have demonstrated that the grb <dig> adaptor acts as a negative modulator of ir kinase activity .
ramos et al.
<cit>  elucidated the mechanism by which the sh <dig> and binding phosphorylated substrate  domains of grb <dig> might induce proteasomal degradation of the ir and subsequent inhibition of its downstream signaling pathways.
the question arises as to whether the adaptor or kinases themselves are regulated and if so, how?
our data demonstrate that there is a reciprocal regulation of rtk phosphorylation activity and expression in the presence of kv <dig> .
holmes et al.
<cit>  similarly reported that treatments that induce membrane hyperpolarization  markedly attenuate tyrosine phosphorylation catalyzed by cellular tyrosine kinases.
unlike their report, centralizing on reciprocal downregulation of kv <dig>  and cellular tks, our data demonstrate that both trkb and ir kinase protein levels are reduced when co-expressed with kv <dig> , which then in turn, decreases the total amount of kinase phosphorylation.
at the same time, we also observe that both nshc and grb <dig> adaptor protein expression is reduced when co-expressed with kv <dig>  , and that the expression of both adaptors is increased in kv <dig> -null mice  <cit> , offering another tier of regulation of the channel/kinase/adaptor interaction involving receptor-linked as opposed to cellular tyrosine kinases.
another suggested method in which grb <dig> action might be terminated is via phosphorylation; in its inactive state it is thought to form tetramers  <cit> .
although we did not test whether grb <dig> was phosphorylated in channel/kinase/adaptor transfected conditions under bdnf-stimulated conditions , this certainly did not functionally affect downregulation of channel expression.
grb <dig> appears to be promiscuous in interacting with multiple receptor and non-receptor tks and other signaling proteins in vitro  <cit> , therefore, gene-targeted deletion of the adaptor could afford physiological relevance to many of these interactions.
grb10-null mice have disproportionate overgrowth of the embryo and placenta, imbalanced glucose homeostasis, and are approximately thirty percent larger at birth  <cit> .
this is an interesting phenotype given the fact that kv <dig> -null mice, oppositely, are resistant to diet-induced obesity, are smaller and physiologically thinner than age-matched wild-type animals, and have increased mobility and metabolism  <cit>  – while having an increased expression of grb <dig> protein that reinforces its function as a growth inhibitor  <cit> .
in heterologous expression systems and in vivo, shc adaptor protein can bind directly to the channel .
shc can also bind to trkb at y <dig> to increase phosphorylation of kv <dig>  and relieve bdnf-induced current suppression.
to what extent is this mimicked in native olfactory bulb neurons ?
we know that exogenous application of bdnf to native obns  <cit> , where shc and trkb are both present  <cit> , causes an increase in kv <dig>  tyrosine phosphorylation and current suppression that is significantly greater than what we observe in heterologous expression systems  <cit>   to 40%  of mitral cell outward current is suppressed by acute bdnf stimulation).
we also know that rtks explicitly target kv <dig> , because in mice with kv <dig>  gene-targeted deletion, neither insulin nor bdnf has any affect on mitral cell current.
thus, there is a divergence in gain strength between hek  <dig> cells and ob neurons for neurotrophic factor signaling that might lie at a missing adaptor protein not mimicked in the hek  <dig> cell system, a lesser abundance of inhibitory adaptor proteins, or perhaps another regulatory molecule as yet not accounted for.
CONCLUSIONS
in native ob, adaptor proteins may change with developmental profile, regeneration, or disease  <cit>  – hence expression patterns of co-localization may be non-static, which would result in state-dependent channel properties.
for example, it is well documented that neurotrophins are altered with injury  <cit>  and that insulin is modified with metabolic state and meals  <cit> , while both are modified with growth or development  <cit> .
therefore, the conductance through a kv <dig>  channel, which underlies the shape and frequency of the action potential output of the ob, would inherently be modified by dynamic change in physiological state.
this in turn might predict that odor perception is very state dependent as dictated by its microenvironment of protein partners and adaptors that would influence a major voltage-gated ion channel in this system.

 voltage-dependent potassium channels are regulators of neuronal excitability. the channels are responsible for maintaining the resting potential of cells, they determine the width and maximum amplitude of the action potential, and they govern the interpulse interval or timing patterns of firing in order to relay sensory information to the brain or coordinate motor output. as recently reviewed by kaczmarek, kv channels may lead a "double life" by possessing non-conducting functions, which allow them to participate in coupled biochemical reactions or communicate directly with cytoplasmic and nuclear signaling pathways. kv, a member of the shaker subfamily of kv channels, is particularly well poised to participate in multiple cell signaling pathways given a number of molecular motifs that serve as protein-protein interaction domains in the n and c terminal aspects of the channel protein. multiple and different combinations of tyrosine residues become phosphorylated upon activation of cellular and receptor tyrosine kinase signaling cascades to elicit changes in ion channel current magnitude, inactivation kinetics, or cumulative/use-dependent inactivation. further levels of complexity exist, however, because the channel is not just a substrate for tyrosine phosphorylation. the phosphorylated tyrosine residue now become recognition motifs for a variety of src homology domain containing proteins that are down-stream signaling molecules, or adaptor proteins, without catalytic activity, and which functionally subserve to "modulate the modulation". two proline-rich domains have been shown to serve as phosphorylation-independent binding domains for src homology containing proteins and ubiquitin ligases to change kv channel clustering and surface expression. gene-targeted deletion of kv demonstrates that the channel's associated scaffold of kinases and adaptor proteins becomes unbalanced in the absence of the channel gene, whereby there is a significant increase in the expression of these modulatory cell signaling proteins, including neurotrophin receptor tyrosine kinase b, the cellular kinase src, the adaptor proteins 14-3- neuronal src homology and collagen, postsynaptic density protein-, and the growth factor receptor-bound protein. because kv protein has an abundance of molecular modules for protein-protein interactions, it is not surprising to discover a diversity of non-conducting functions following targeted deletion of the core of the scaffold. this study focuses upon two adaptor proteins enriched in the olfactory bulb, namely grb and nshc. the adaptor protein grb is a member of a superfamily of adaptor proteins that also includes grb grb and mig-. the characterization of conserved molecular architecture of this family has shown that members all contain a c-terminal sh domain, a central segment containing a pleckstrin homology domain, and a principle binding region for association with insulin-like growth factor receptor type that also acts to stabilize insulin receptor kinase binding to the sh domain. the n terminal domain of grb is less well conserved but contains multiple pxxp domains that serve as putative binding sites for sh domain containing proteins. while grb was originally discovered to be an interacting protein with the epidermal growth factor receptor and later found to be a negative regulator of the ir via ubiquitination, it may also interact with other rtks. grb has been linked with human growth retardation and type diabetes when upregulated or mutated, which alters grb protein-protein interactions. the adaptor protein nshc belongs to a family that includes three major genes, shca, shcb, and shcc. all shc isoforms possess two distinct domains that bind phosphotyrosine containing sequences, namely, the phosphotyrosine binding domain and the sh domain, and a central domain that contains multiple tyrosine phosphorylation sites. nshc is specifically enriched in adult brain and has been found to direct survival and phenotypic maturation of neurons via association with neurotrophin receptors. more recently, nshc has been also implicated in the modulation of hippocampal synaptic plasticity via modulation of the nmda receptor, independent of neurotrophin receptor signaling. previous site-directed mutagenesis studies in heterologous expression systems, have demonstrated that bdnf-activation of trkb kinase phosphorylates kv at three tyrosine residues or clusters in the n- and c-termini of the channel, y111– y y to cause suppression of current magnitude without changes in channel inactivation or deactivation kinetics. our current strategy was to co-express channel and kinase with one of two adaptor proteins that varied in their reported interactions: an adaptor protein that was not known to directly associate with the neurotrophin receptor or the channel, and an adaptor protein that was known to bind to y of the neurotrophin receptor. unexpectedly, we discovered that nshc adaptor proteins reportedly characterized to interact with rtks also used kv channel for a binding partner directly. grb did not interact with either the channel or kinase, but could indirectly modulate the channel by changing its surface expression. in native ob, however, both adaptor proteins co-ip with kv and trkb kinase. our data suggest that there is a complex regulation between the channel, the kinase, and adaptor proteins such that the expression of kv down regulates that of trkb kinase, while the expression of grb likely acts to induce ubiquitination of the channel thus decreasing channel surface expression.  co-localization of adaptor proteins and kv channel in the olfactory bulb although the goal of our study was to define the molecular targets for interaction of kv plus trkb kinase in the presence of adaptor proteins using a well-defined heterologous expression system, it was important to ascertain the physiological significance of such interactions by first exploring the potential co-localization of the adaptor proteins with the channel in native brain. kv has a limited distribution in the central nervous system, where it has been studied primarily in regions of highest expression, namely in the olfactory bulb, the olfactory cortex, and the dentate gyrus of the hippocampus. we have previously reported the expression of several adaptor proteins in cytoplasmic lysates of the ob by western blot analysis as well as the co-localization and co-ip of the channel and trkb kinase in the ob and olfactory cortex. thus, we explored co-localization of kv with that of nshc and grb in the ob of postnatal day 20– mice where kv channel properties are well characterized and it is known that modulation of kv alters olfactory threshold. kv and nshc demonstrated greatest overlap of expression in the peripheral structures surrounding the glomeruli and weaker co-localization patterns in the external plexiform layer, the mitral cell layer, the internal plexiform layer, and the granule cell layer. kv and grb demonstrated greatest overlap of expression in the ipl and gcl and weaker co-localization patterns in some fibers surrounding the glomeruli. adaptors can directly co-ip with kv channel independent of trkb kinase since both adaptors demonstrated a degree of colocalization with kv in native brain tissue, we sought more rigorous conformation for a potential protein-protein interaction using hek cells in which site specific mutation of the channel could be performed to map the molecular targets for channel-adaptor scaffolding. we have previously demonstrated that bdnf-induced kv current suppression is mediated through tyrosine phosphorylation of three sites in the n and c termini of the channel, y111– y and y. since both nshc and grb have sh domains that could target any or a combination of these tyrosine residues upon bdnf-induced trkb phosphorylation, we wanted to test whether either adaptor protein could associate with the channel under bdnf-stimulated conditions. as demonstrated in fig. 4a, immunoprecipitation of myc-tagged kv, followed by western analysis and blotting for shc labels predominantly a single kda band in hek cells that were only co-transfected with myckv + shc. therefore, the channel and the shc adaptor protein form a protein-protein interaction complex independent of bdnf-induced phosphorylation of the channel or at least in the absence of trkb kinase. inclusion of trkb kinase or y490f trkb cdna into the transfection scheme does not prevent or increase the channel/adaptor interaction, further exemplifying the lack of requirement of trkb in the channel/adaptor complex. as shown in fig. 4b, we were not able to co-immunoprecipitate trkb and grb or kv and grb, indicating that unlike nshc adaptor protein, grb does not associate with the kinase. also, note the expression of native levels of adaptor proteins, apparently endogenous to hek cells in lanes untransfected with shc or grb cdna. adaptor/kinase/channel scaffold can be precipitated from olfactory bulb and hippocampus given our ability to co-immunoprecipitate nshc and kv channel, we next explored whether the proteins interacted in native tissue by using two regions known to express kv. as demonstrated in fig. immunoprecipitation of kv, followed by western analysis and blotting for anti-trkb, anti-shc, or anti-grb labels protein bands of predicted molecular mass for the kinase, and adaptors respectively. as we have previously reported for the olfactory bulb, trkb labeling demonstrated a greater intensity for the full-length receptor protein over that of its truncated form that lacks a kinase domain. the predicted molecular mass for shc is multiple depending upon the variant and in previous cytosolic fractions we have observed all three variants in olfactory bulb preparations. it is interesting that in the hek cells, the molecular migration was closest to the kda variant, whereas in the olfactory bulb, the variant associated with kv was different than those associated with trkb. moving to the hippocampus, the variants forming a protein-protein interaction with kv were also different than that in the olfactory bulb. it is also noteworthy, that while grb could not be consistently co-immunoprecipitated with either the kinase nor the channel via heterologous expression, both were complexed with the adaptor protein in the olfactory bulb and the hippocampus. it is possible in the hek cells that the interaction is at the limit of our detection. it may also suggest either a a missing scaffolding component in the hek cells that is essential for protein-protein interactions with the grb adaptor or a different regulation of grb in neurons. grb decreases surface expression density of the channel we have previously reported that kv channel modulation can be perturbed by adaptor proteins via disruption of src- or insulin-induced kv phosphorylation. in the cellular tyrosine kinase signaling pathway, adaptor proteins that do not disrupt tyrosine phosphorylation have no effect on channel function; however, those that decrease tyrosine phosphorylation can relieve kinase-induced current suppression. in the receptor tyrosine kinase signaling pathway, adaptor proteins that disrupt tyrosine phosphorylation relieve both current suppression and simultaneously act to increase the rate of channel inactivation. for the neurotrophin pathway, we questioned how the degree of channel phosphorylation as modified by an adaptor protein might be linked to function. thus, we immunoprecipitated kv channel plus trkb kinase under various transfection conditions that included either shc or grb adaptors. interestingly, inclusion of nshc in the transfection scheme increased the y phosphorylation of the channel over that observed under the co-transfection condition alone. moreover, elimination of the y trkb site via substitution with trkbshc- in the channel/kinase/adaptor transfection scheme completely prevented tyrosine phosphorylation of the channel. basal phosphorylation of kv was higher than expected or reported in our previous studies, but may be attributed to the solution used as a vehicle carrier that may have slightly depolarized the cells. none the less, all basal phosphorylation could be completely eliminated in the absence of trkb or via substitution with a dead trkb kinase construct in which the catalytic domain was truncated. most interestingly, inclusion of grb in the transfection scheme not only completely eliminated the y phosphorylation of the channel over that observed in the co-transfection condition, blotting the lysates with anti-kv to control for expression of the channel, revealed that grb was greatly reducing the expression of kv. the degree of bdnf-induced kv phosphorylation in each transfection condition was quantified by densitometry, normalized to that of the kv channel transfection alone, and statistically compared in the bar plot of fig. 6c. expression levels of grb kv, and trkb are cross-regulated to further explore the robustness of our finding of grb downregulation of kv, we compared the surface fraction of kv channels and the total kv pool in the presence and absence of grb as shown in fig. 7a, b both the surface fraction of kv channels as well as the total pool of kv is dramatically down regulated by grb adaptor protein. grb also had the capacity to down regulate kv protein expression in the presence of trkb kinase. in conjunction with the ability to downregulate total channel protein, immunocytochemical experiments demonstrated a significant reduction in fluorescent signal at the membrane under grb co-transfection conditions. albeit lack of grb10/kv co-immunoprecipitation in vitro, if grb was downregulating the expression of kv channel via the sh recognition domains on kv, then substitution of y to f mutations in the n- and c-terminal aspects of the channel should completely eliminate any reduction in expression. single point mutagenesis at the selected y residues of this study does not alter expression of the derived channel mutants as determined by either western analysis or electrophysiology. western analysis of hek cells in which grb was co-transfected with wild-type or various y to f channel mutants revealed that residues yyy111– and y represent important residues for regulation between channel and adaptor protein. removal of these residues prevented kv downregulation by grb co-transfection. mutation of residues y and y retained the trend for down-regulation of kv, thus these residues are likely not involved in the adaptor's regulation of channel expression. next, we tested whether channel conductance was necessary for grb down-regulation of kv by substituting the pore-conducting mutant w386f kv. as shown in fig. 7d and 7e, mutation of the pore had no effect in the adaptor's ability to down-regulate kv channel expression. finally, we tested whether the grb down-regulation of kv was specific to this particular shaker subfamily member or could be extended to other potassium channels. substitution in the transfection scheme of either kv or kv, two family members that are also expressed in olfactory bulb neurons, elicited no change in channel expression when co-transfected with grb. furthermore, expression of general structural proteins also demonstrated no change in expression in conditions of grb transfection. adaptor proteins grb and nshc, respectively decreased and increased the expression of kv channel. of noteworthy regulation, curiously it was observed that co-transfection with kv channel protein decreased both the expression of trkb as well as the phosphorylated state of the kinase when stimulated with bdnf. this was not only true for trkb, but also was observed for a different, receptor tyrosine kinase. expression of kv channel also decreased the expression of ir kinase protein and its concomitant phosphorylation by insulin. the degree of kv expression in the presence and absence of trkb or ir kinase was quantified by densitometry, normalized to that of the kv channel transfection alone, and statistically compared in the bar plot of fig. 8b. these data pose a complex balance between channel, kinase, and adaptor whereby the level of an adaptor protein could down or upregulate expression of the channel, which could in turn, dial in the degree of phosphorylation of the kinase. bdnf-stimulated channel modulation is blocked by grb and nshc adaptor proteins if the two adaptor proteins differentially co-immunoprecipitated with kv channel, one might predict a functional difference in their ability to modulate the channel as determined through standard patch-clamp electrophysiology. hek cells were thus transiently transfected with kv plus trkb kinase in the presence or absence of nshc or grb adaptor protein and monitored for modulation of biophysical properties during a twenty minute acute stimulation with brain-derived neurotrophic factor. approximately hours post-transfection, macroscopic outward currents were recorded in the cell-attached configuration. cells were voltage-clamped at - mv and stepped to a depolarizing potential of + mv for second to activate kv. table reports kv peak current amplitude, inactivation time constant, deactivation time constant, voltage at half-activation, and voltage dependence for a population of recorded patches under various transfection conditions pre and post growth factor application. twenty minute application of bdnf had no effect on patches transfected with kv channel alone. this control confirms that application of ligand alone in the absence of the receptor fails to modulate kv. inclusion of kv plus trkb significantly increased peak current amplitude without altering kinetic properties. peak current amplitude was +/- pa in the kv -only control transfection condition and +/- pa in the kv plus trkb kinase condition, confirming a statistically significant 90% increase in kv current in the presence of the kinase. we have previously reported that trkb kinase co-expression in the absence of bdnf stimulation increases the channel half-life at the plasma membrane so the increase in kv current was as expected. secondarily, these records demonstrate typical stability of the patch over the recording interval for the co-transfection condition. inclusion of ng/μl bdnf in the patch electrode evoked a significant suppression of peak current amplitude of kv to 85% of its original current that was relieved by co-transfection of either adaptor protein. in addition, we noted that initial peak current amplitude of kv in the co-transfection condition ) was significantly less in the presence of nshc ) but was unchanged in the presence of grb ). regardless of the pre-stimulation peak current, inclusion of either adaptor protein, prevented bdnf-induced kv current suppression. fig. 9e is a histogram representation of the normalized bdnf-induced kv current suppression ) that is relieved in the presence of shc or grb. for grb the relief in bdnf-induced current suppression appeared to be coincident with a slight shift in kv voltage dependence. there was a to mv left shift in the voltage at half-maximum activation in the presence of trkb plus grb as calculated from a boltzman fit of the tail currents when cells were held at - mv and stepped to + mv in increments of mv using a pulse duration of ms. because this was not a robust effect and was somewhat variable even in the absence of an adaptor protein, this biophysical property was not further studied. hek cells were transfected with cdnas encoding kv ± trkb ki nase ± adaptor protein. several voltage-stimulating paradigms were completed to collect the tabled biophysical properties by cell-attached patch-clamp recording. mean values for peak current amplitude, inactivation time constant, deactivation time constant, voltage at half activation, and slope of the voltage dependence were compared across various transfection conditions at the start of the patch recording versus twenty minutes following ng/μg bdnf using a paired t-test, α ≤. * = significantly different. nshc signaling at y trkb is target for blocked modulation of kv channel to our knowledge there no reported direct interactions between trkb and grb so we focused our study upon disruption of known targets of shc binding to trkb kinase. the sh domain of nshc is known to bind phosphorylated y trkb at the npqpy recognition motif. if the molecular target for nshc perturbation of bdnf-induced kv current suppression was soley y on trkb kinase, then destruction of this binding site should regain bdnf modulation of kv. what we found by substituting the trkb kinase mutant construct, however, was an intermediate effect. co-transfection of kv + trkbshc- plus nshc cdna exhibited bdnf-induced current suppression of kv versus +/- pa that did not reach statistical significance compared with that observed with kv + trkb co-expression versus +/- pa; significantly different paired t-test, α ≤; n= 84% suppression). if, however, we substituted grb in the transfection scheme instead of nshc, we observed retention of bdnf-induced current suppression that was not relieved by the adaptor protein versus +/- pa; significantly different paired t-test, α ≤; n = 71% suppression). although grb has never been reported to directly interact with trkb kinase signaling cascades, and we also do not find that this adaptor co-immunoprecipitates with the kinase in vitro, residue y of trkb kinase is an important target for nshc, and its mutation appears to somehow uncouple grb modulation of the channel. with the removal of y of trkb kinase, grb can no longer relieve bdnf-induced current suppression of the channel. interestingly, although neither adaptor protein induces a change in peak current amplitude of kv alone, grb also evoked a faster rate of channel inactivation versus +/- msec as demonstrated in fig. 10e and statistically compared in table hek cels were transfected with cdnas encoding kv +trkb kinase or trkbshc- ± adaptor protein. several voltage-stimulating paradigms were completed to collect the tabled biophysical properties by cell-attached patch-clamp recording. bdnf stimulation, notations, and statistical analysis as in table discussion kv is a substrate for multiple tyrosine kinase signaling cascades including ir, trkb, and src kinase. each exerts the phosphorylation of multiple tyrosine residues, and different combinations of residues, to produce a unified response, of kv current suppression. inclusion of adaptor proteins causes perturbation of the channel current suppression, and in the case of neurotrophin signaling, we find that disruption of the channel modulation is not directly correlated to the extent of channel phosphorylation. the scenario is more complex. this may be attributed to the fact that the interaction between kinase and channel is no longer a binary relationship, with the added adaptor having the capacity to bind to both channel and kinase to elicit different responses. not only can an adaptor protein alter how the channel will respond to neurotrophin-induced current suppression, the adaptors alter expression levels of the channel and the channel itself can downregulate trkb expression and its phosphorylated state. such a dynamic equilibrium between kv /trkb/adaptors may allow a full spectrum of modulation of channel properties dependent upon the compliment of proteins co-expressed in a neuron at a given physiological state. biochemically, the degree of channel phosphorylation by bdnf-induced trkb activation is no longer directly related to the degree of functional current suppression when adaptors are added to the mix. this is very different than what is reported for the cellular tyrosine kinase, src. here, phosphorylation was directly linked to the degree of current suppression and one could predict the modulation of the selected adaptor protein based upon how it influenced tyrosine phosphorylation of the channel. this is in stark contrast to rtks. the addition of nshc to kv plus trkb transfected conditions induced an increase in both channel expression as well as bdnf-induced channel phosphorylation whereas the addition of grb resulted in the opposite. despite the fact that the adaptors evoke an opposite response biochemically, the functional ramification is identical, the prevention of bdnf-induced current suppression. in fact, nshc/grb have structurally similar sh domains whereas psd- lacks such phosphorylation-dependent interactive domains, and yet each has the capacity to functionally perturb the bdnf-induced current suppression. this might suggest that direct interaction of the adaptor and the channel may play a predominant role in preventing kinase modulation and that changed phosphorylation is a secondary effect. in the case of the two sh domain containing adaptor proteins, nshc and grb binding of y trkb by nshc is an additional key regulatory site impacting modulation of the channel. certainly there are endogenous levels of both nshc and grb expressed in hek cells, thus our kv + trkb conditions are not completed devoid of adaptor protein, however, when nshc is co-transfected with these conditions, binding of y trkb induces a block of bdnf-induced channel suppression. kv channel expression is changed in the presence of trkb kinase without bdnf stimulation and now we can also see that grb adaptor significantly modifies surface expression of kv. with trkb kinase co-expression, there is a correlate increase in channel surface expression, increase in channel protein expression, and increase in channel macroscopic current. interestingly, with grb co-expression there is oppositely a decrease in channel surface expression, a decrease in protein expression of the channel, but no change in channel macroscopic current, rather an increased rate of channel inactivation. the adaptor/channel do not form a complex in vitro that we can detect, nevertheless, co-expression apparently changes surface distribution and channel density but not current magnitude or conduction. this would imply that there must be many kv channels that are not contributing to current. such a pool of silent or "sleeping channels" has been reported for specific kv subfamily members and uncovered for other potassium channels during frac. our data and that reported by sun et al. support that molecular/protein density of a channel may not always exhibit linearity with functional density. based upon the crystal structure of grb sh domain determined at Å resolution, it is suggested that binding of grb is dimeric, using turn containing phosphotyrosine sequences. it is unknown if the kv channel contains turn containing phosphotyrosine motifs unless the tetrameric structure of the subunits permitted such an alignment, which would be contrary to positioning of the α transmembrane helices and for kv, a shaker family member for which crystal structure has been reported. on the basis of structural analysis and previous biochemical studies for the adaptor, grb and grb have a partially impaired ability to bind phosphotyrosine-containing ligands due to a non-glycyl residue at the end of the bd loop and lack of a p + binding pocket. these combined data are consistent with our findings that we cannot detect grb and kv co-immunoprecipition in vitro, albeit the fact that channel/adaptor functional interactions appear to modulate channel expression and can be found as a complex in unstimulated native tissues. the fact that mutation of several y residues on the n and c terminal aspects of the channel clearly perturbs the negative modulatory affect of grb on channel expression, indicates a specific modulatory role that does not require tight association. moreover, the modulation does not require channel activity, or at least functional ion conductance. while all y to f channel mutations blocked grb down regulation of kv expression, yyy111-113fff kv and y449f kv appear to be the most robust at blocking down regulation. both of these sites were previously reported as targets for bdnf-induced kv current suppression and phosphorylation but only y has a large hydrophobic m downstream of the phosphotyrosine, reported as a preferred grb motif. it has previously been reported that the e ubiquitin protein ligase called nedd4- regulates voltage-gated ion channels, including nav, kir, and kv family members via targeting phosphotyrosine motifs located in the c-terminal aspect of the channel. it has also been demonstrated that the sh domains of grb form a constitutive complex with nedd4- so as to mediate ligand-dependent ubiquitination of igf-ir. since co-expression of nedd4- with kv in xenopus oocytes induces a strong suppression of kv current we conjecture that grb might bind to endogenous nedd4- in our system to cause ubiquitination of kv and/or trkb kinase. this might explain a potential mechanism for decreased kv surface expression in the presence of grb and correlate decreased channel tyrosine phosphorylation. we have shown that trkb and grb act as modulators of channel expression, while others have demonstrated that the grb adaptor acts as a negative modulator of ir kinase activity. ramos et al. elucidated the mechanism by which the sh and binding phosphorylated substrate domains of grb might induce proteasomal degradation of the ir and subsequent inhibition of its downstream signaling pathways. the question arises as to whether the adaptor or kinases themselves are regulated and if so, how? our data demonstrate that there is a reciprocal regulation of rtk phosphorylation activity and expression in the presence of kv. holmes et al. similarly reported that treatments that induce membrane hyperpolarization markedly attenuate tyrosine phosphorylation catalyzed by cellular tyrosine kinases. unlike their report, centralizing on reciprocal downregulation of kv and cellular tks, our data demonstrate that both trkb and ir kinase protein levels are reduced when co-expressed with kv, which then in turn, decreases the total amount of kinase phosphorylation. at the same time, we also observe that both nshc and grb adaptor protein expression is reduced when co-expressed with kv, and that the expression of both adaptors is increased in kv -null mice, offering another tier of regulation of the channel/kinase/adaptor interaction involving receptor-linked as opposed to cellular tyrosine kinases. another suggested method in which grb action might be terminated is via phosphorylation; in its inactive state it is thought to form tetramers. although we did not test whether grb was phosphorylated in channel/kinase/adaptor transfected conditions under bdnf-stimulated conditions, this certainly did not functionally affect downregulation of channel expression. grb appears to be promiscuous in interacting with multiple receptor and non-receptor tks and other signaling proteins in vitro, therefore, gene-targeted deletion of the adaptor could afford physiological relevance to many of these interactions. grb10-null mice have disproportionate overgrowth of the embryo and placenta, imbalanced glucose homeostasis, and are approximately thirty percent larger at birth. this is an interesting phenotype given the fact that kv -null mice, oppositely, are resistant to diet-induced obesity, are smaller and physiologically thinner than age-matched wild-type animals, and have increased mobility and metabolism – while having an increased expression of grb protein that reinforces its function as a growth inhibitor. in heterologous expression systems and in vivo, shc adaptor protein can bind directly to the channel. shc can also bind to trkb at y to increase phosphorylation of kv and relieve bdnf-induced current suppression. to what extent is this mimicked in native olfactory bulb neurons? we know that exogenous application of bdnf to native obns, where shc and trkb are both present, causes an increase in kv tyrosine phosphorylation and current suppression that is significantly greater than what we observe in heterologous expression systems to 40% of mitral cell outward current is suppressed by acute bdnf stimulation). we also know that rtks explicitly target kv, because in mice with kv gene-targeted deletion, neither insulin nor bdnf has any affect on mitral cell current. thus, there is a divergence in gain strength between hek cells and ob neurons for neurotrophic factor signaling that might lie at a missing adaptor protein not mimicked in the hek cell system, a lesser abundance of inhibitory adaptor proteins, or perhaps another regulatory molecule as yet not accounted for.  in native ob, adaptor proteins may change with developmental profile, regeneration, or disease – hence expression patterns of co-localization may be non-static, which would result in state-dependent channel properties. for example, it is well documented that neurotrophins are altered with injury and that insulin is modified with metabolic state and meals, while both are modified with growth or development. therefore, the conductance through a kv channel, which underlies the shape and frequency of the action potential output of the ob, would inherently be modified by dynamic change in physiological state. this in turn might predict that odor perception is very state dependent as dictated by its microenvironment of protein partners and adaptors that would influence a major voltage-gated ion channel in this system. 
previously we have shown that acute brain-derived neurotrophic factor activation of neurotrophin receptor tyrosine kinase b suppresses the shaker voltage-gated potassium channel via phosphorylation of multiple tyrosine residues in the n and c terminal aspects of the channel protein. it is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity. we report the co-localization of two adaptor proteins, neuronal src homology and collagen and growth factor receptor-binding protein, with kv channel as demonstrated through immunocytochemical approaches in the olfactory bulb neural lamina. in the absence of either adaptor protein, channel co-expression reciprocally down-regulated expression and tyrosine phosphorylation of trkb kinase and related insulin receptor kinase. we report that adaptor protein alteration of kinase-induced kv channel modulation is related to the degree of direct protein-protein association and that the channel itself can reciprocally modulate receptor-linked tyrosine kinase expression and activity. to examine the possibility that the src homology domains of grb were directly binding to basally phosphorylated tyrosines in kv, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. finally, through patch-clamp electrophysiology, we found that the bdnf-induced current suppression of the channel was prevented by both nshc and grb to further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and western analysis of channel, kinase, and adaptor transfected human embryonic kidney cells. both adaptors, however, co-immunoprecipitated with kv in native ob.
 neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. previously we have shown that acute brain-derived neurotrophic factor activation of neurotrophin receptor tyrosine kinase b suppresses the shaker voltage-gated potassium channel via phosphorylation of multiple tyrosine residues in the n and c terminal aspects of the channel protein. it is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity.  we report the co-localization of two adaptor proteins, neuronal src homology and collagen and growth factor receptor-binding protein, with kv channel as demonstrated through immunocytochemical approaches in the olfactory bulb neural lamina. to further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and western analysis of channel, kinase, and adaptor transfected human embryonic kidney cells. nshc formed a direct protein-protein interaction with kv that was independent of bdnf-induced phosphorylation of kv, whereas grb did not complex with kv in hek cells. both adaptors, however, co-immunoprecipitated with kv in native ob. grb was interestingly able to decrease the total expression of kv, particularly at the membrane surface, and subsequently eliminated the bdnf-induced phosphorylation of kv. to examine the possibility that the src homology domains of grb were directly binding to basally phosphorylated tyrosines in kv, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. removal of the tyrosines 111– and prevented grb from decreasing kv expression. in the absence of either adaptor protein, channel co-expression reciprocally down-regulated expression and tyrosine phosphorylation of trkb kinase and related insulin receptor kinase. finally, through patch-clamp electrophysiology, we found that the bdnf-induced current suppression of the channel was prevented by both nshc and grb  we report that adaptor protein alteration of kinase-induced kv channel modulation is related to the degree of direct protein-protein association and that the channel itself can reciprocally modulate receptor-linked tyrosine kinase expression and activity. 