Two adaptor proteins differentially modulate the phosphorylation and biophysics of Kv1.3 ion channel by Src kinase

Solutions and Reagents

Human embryonic kidney cell (HEK 293) patch pipette solution contained (in mM): 30 KCl, 120 NaCl, 10 HEPES, and 2 CaCl2 (pH 7.4). HEK 293 cell recording bath solution contained (in mM): 150 KCl, 10 HEPES, 1 EGTA, and 0.5 MgCl2 (pH 7.4). Cell lysis buffer with protease and phosphatase inhibitors (PPI solution) contained (in mM): 25 Tris (hydroxymethyl) aminomethane (pH 7.5), 250 NaCl, 5 EDTA, 1% Triton X-100, 1 sodium orthovanadate, 1:100 dilution of Sigma Phosphatase Inhibitor Cocktail II (Catalog # P-5726), 1:500 dilution of Sigma Protease Inhibitor Cocktail (Catalog # P-8340). Wash buffer contained (in mM): 25 Tris (pH 7.5), 250 NaCl, 5 EDTA, and 0.1% Triton X-100. Homogenization buffer contained (in mM): 320 sucrose, 10 Tris base, 50 KCl, and 1 EDTA (pH 7.8). All salts were purchased from Sigma Chemical Company or Fisher Scientific. Tissue culture and transfection reagents were purchased from Gibco BRL.

Olfactory Bulb Membrane Solubilization

One set of olfactory bulbs (OB) from Sprague-Dawley rats at postnatal day 17 (P17) and P30 (adult) were quickly dissected using American Veterinary Medical Association (AVMA)- and National Institutes of Health (NIH)-approved methods. Harvested OB tissue was homogenized on ice using a Kontes tissue grinder (size 20; 50 strokes) and homogenization buffer containing 0.5% nonidet P-40 (NP-40) detergent and Sigma protease and phosphatase inhibitor cocktails (see Solutions and Reagents). The mixture was vortexed every 15 minutes for 4 hours, centrifuged at 14,000 × g for 10 minutes at 4°C, and the supernatant (lysate) was stored at -80 °C until subsequent use. Protein concentration was determined by Bradford protein assay; 10 or 40 mg of lysate was separated by 10% SDS-PAGE, proteins were electrotransferred to nitrocellulose, and Western blots were probed with antisera for Src, Shc, and Grb (see below).

cDNA Constructs and Antibodies

All channel, kinase, and adaptor protein coding regions were downstream from a cytomegalovirus (CMV) promoter. The v-Src cDNA construct was a generous gift from Dr. Richard Huganir (Johns Hopkins University, Baltimore, MD) and was located in a modified PRK5 vector. The R385A v-Src cDNA construct (v-Src with severely impaired tyrosine kinase activity) was a generous gift from Dr. M. Senften (Friedrich Miescher-Institute, Basel Switzerland; {⁴⁰}). The entire R385A v-Src coding region was inserted into a modified PRK5 vector {⁴¹}. Neuronal Shc (n-Shc), Sck, and n-Shc mutant cDNA (Y to F point mutations at tyr²²⁰, tyr²²¹, tyr³⁰⁴) were generous gifts from Dr. T. Nakamura (Sumitomo Electric Industries, Yokohama, Japan) and were expressed in the vector pCMV1 {³²}. The n-shc mutant will be referred to as the “triple”mutant. Grb10 cDNA was a gift from Dr. R. Roth (Stanford University) and was expressed in pBlueScript SK (-) vector (Stratagene).

AU13, a rabbit polyclonal Kv1.3 antiserum, was designed against the 46-amino acid sequence (478MVIEEGGMNHSAFPQTPFKTGNSTATCTTNNNPNDCVNIKKIFTDV523) representing the unique coding region of Kv1.3 between the carboxyl terminus and transmembrane domain 1. The purified peptide was produced by Genmed Synthesis (San Francisco, CA), and the antiserum was generated by Cocalico Biologicals (Reamstown, PA). This antibody was used for immunoprecipitation (1:1000) and Western blot detection (1:1500) of Kv1.3 as in Tucker and Fadool (submitted). The monoclonal antibody 4G10 (Upstate Biochemical) recognized phosphorylated tyrosine residues and was used at 3.5 μg/ml for immunoprecipitation and 1:1000 for Western analysis. Src monoclonal antibody, directed against the Src SH3 domain (Oncogene Ab-1), was used at 10 μg/ml for immunoprecipitation and 1:40-1:100 for Western analysis. Polyclonal antisera for Shc (H-108) and Grb10 (K-20) were from Santa Cruz; each was used at 20 μg/ml for immunoprecipitation and between 1:200-1000 for Western blot detection.

Maintenance of HEK 293 Cells and Transfection

HEK 293 cells were maintained in Minimum Essential Medium (MEM), 2% penicillin/streptomycin, and 10% FBS (Gibco BRL). Before transfection, cells were grown to 100% confluency (7 days), dissociated with trypsin-EDTA (Sigma) and mechanical trituration, diluted in MEM to a concentration of 600 cells/ml, and replated on Corning dishes (Catalog # 25000, Fisher Scientific). cDNA was introduced into HEK 293 cells with a lipofectamine reagent (Gibco BRL) 3-5 days after passage as previously described {^12,13}. Briefly, cells were transfected for 4-5 hours with 0.5-0.75 μg of each cDNA construct per 35-mm dish for electrophysiology or 3.5-5.0 μg of each cDNA construct per 60-mm dish for biochemistry. Plasmid DNA with no coding insert (control vector) served as the control to equalize total μg of cDNA added to each dish. Cells were either harvested for biochemical analysis or used for electrophysiological recordings approximately 30-40 hours after transfection.

For patch-clamp experiments, transfection efficiency was monitored by co-transfecting with pHook (Invitrogen) as a means of rapidly selecting cells expressing Kv1.3 channels. pHook encodes a transmembrane domain from the platelet-derived growth factor receptor (PDGF-R), which is then anchored on the extracellular side of the plasma membrane. Before patch recording, a brief incubation with an appropriate antibody linked to a 5-μm polystyrene bead allowed recognition of transfected cells. Efficiency of co-transfection (greater than 95%) with our transfection method has been tested using double, sequential labeling and confocal microscopic visualization, as reported previously{¹³}.

Electrophysiology

Patch electrodes were fabricated from Jencons glass (Jencons Limited, Befordshire, UK), fire-polished to approximately 1 μm, and coated near the tip with beeswax to reduce the electrode capacitance. Pipette resistances were between 9 and 14 MΩ. Hoffman modulation contrast optics was used to visualize cells at 40× magnification (Axiovert 135, Carl Zeiss). Macroscopic currents in cell-attached membrane patches were recorded using an Axopatch-200B amplifier (Axon Instruments), filtered at 2 kHz, digitized at 2-5 kHz, and stored for later analysis. All voltage signals were generated and data were acquired with the use of either a Microstar DAP 800/2 board with in house written software (DapClamp; Microstar Lab, Bellevue, WA) or an Axon Digidata 1200 board with pClamp software (Axon Instruments). Data were analyzed using Microcal Origin software.

Outward macroscopic currents were recorded in the cell-attached rather than whole-cell configuration; Kv1.3 channel expression is so robust in the HEK 293 expression system that it is not routinely possible to record whole-cell currents without saturating the amplifier {^41-43}. Patches were held routinely at a holding potential of -90 mV and stepped in 20 mV depolarizing potentials using a pulse duration of 1000 milliseconds. Pulses were generally delivered at intervals of 60 seconds or longer to prevent cumulative inactivation of the Kv1.3 channel {⁴⁴}. Kv1.3 peak current amplitude, channel inactivation (τ_inact) and deactivation (τ_deact) kinetics, voltage at one-half maximal activation (V_1/2), and slope of voltage dependence (κ) were measured in the presence of Src kinase and the presence or absence of an adaptor protein (N-Shc or Grb10). Each biophysical property was analyzed in the form of non-normalized data by one-way ANOVA with a Student Newman Keuls (snk) follow-up test at the 95% confidence level to determine any statistical difference in Kv1.3 channel function in the presence of kinase or adaptor proteins. For graphical representations of peak current amplitudes among different transfection conditions, measurements were normalized to the transfection condition of Kv1.3 alone within a single recording session and respective transfection set. Kinetic properties of Kv1.3 have been reported to be independent of current magnitude {⁴⁴}, thus peak current amplitude but not kinetic data were normalized to adjust for differences in channel expression between transfections.

Fitting parameters for inactivation and deactivation kinetics were as previously described {¹³}. Briefly, the inactivation of the macroscopic current, during a 1000 ms voltage step from -90 to +40 mV, was fit to the sum of two exponentials by minimizing the sums of squares using a bi-exponential function (y = y₀ + A₁e^-(x-x0)/τ1 + A₂e^-(x-x0)/τ2). The two inactivation time constants (τ₁ and τ₂) were combined by multiplying each by its weight (A) and summing. The deactivation of the macroscopic current was fit similarly but to a single exponential (y = y₀ + Ae^-(x-x0)/τ). Tail current amplitudes were plotted in a current-voltage relationship and fit to a Boltzmann sigmoidal curve (Y = [(A₁ + A₂)/(1 + e^(x-x0)/dx)] + A₂) to calculate the slope of voltage dependence (κ) and voltage at half-activation (V_1/2) for Kv1.3. To study cumulative inactivation of Kv1.3 under the control condition and in the presence of Src and/or an adaptor protein, patches were held at -90 mV and stepped to +40 mV twelve times using a similar pulse duration as above but recording using different interpulse intervals, including 60, 30, 10, 2, 1, and 0.5 seconds. Peak current amplitudes were normalized to the initial trace in each series for a given interpulse interval. Cumulative inactivation was always present with interpulse intervals less than 60 seconds in the control (Kv1.3 alone) transfection condition, and as much as 50% of channels were caught in the inactivated state with repeated stimulation less than 2 seconds.

Immunoprecipitation and Electrophoretic Separation

Transfected cells were harvested by lysis 2 days post-transfection in ice-cold PPI solution (see Solutions and Reagents). The lysates were clarified by centrifugation at 14,000 × g for 10 minutes at 4°C and incubated for 1 hour (h) with 0.2-0.3 mg/ml protein A-sepharose (Amersham-Pharmacia), followed by re-centrifugation to remove the protein A-sepharose. To test the phosphorylation state of Kv1.3 in the presence of v-Src kinase plus or minus an adaptor protein, tyrosine-phosphorylated proteins were immunoprecipitated from the clarified lysate by overnight incubation at 4°C with 3-4 μg/ml 4G10 antibody (Upstate Biochemical). Immunoprecipitated proteins were harvested by a 2-h incubation with protein A-sepharose and centrifugation as above. The immunoprecipitates (IPs) were washed 3 times with ice-cold wash buffer. Lysates and washed IPs were diluted in sodium dodecyl sulfate (SDS) gel loading buffer {⁴⁵} containing 1 mM Na₃VO₄. Protein concentration was determined by Bradford protein assay. Proteins were separated on 10% acrylamide SDS gels and electrophoretically transferred to nitrocellulose for Western blot analysis. Nitrocellulose membranes were blocked with 5% nonfat milk and incubated overnight at 4°C in primary antibody against Kv1.3, v-Src, Shc, or Grb10. Membranes were then exposed to species-specific peroxidase-conjugated secondary (Amersham-Pharmacia or Sigma) for 90 minutes at room temperature. Enhanced chemiluminescence (ECL; Amersham-Pharmacia) exposure of Fuji RX film (Fisher) was used to visualize labeled protein.

Resultant bands were quantified by densitometry using a Hewlett-Packard Photosmart Scanner in conjunction with Quantiscan software (Biosoft, Cambridge, UK). Pixel densities for each band were normalized to Kv1.3 within the same cell passage, transfection, and autoradiograph. Mean pixel densities were then calculated across sets of normalized data contained in single autoradiographs. This type of quantification was designed to reduce inherent variability in cell culture, transient transfection efficiencies, and ECL exposure times. Statistical significance was set at the 95% confidence level for immunodensitometry data that were analyzed by one-way ANOVA with snk follow-up test.

Signaling Proteins are Expressed in the Olfactory Bulb

Kv1.3 becomes robustly phosphorylated at tyr¹³⁷ and tyr⁴⁴⁹ when co-expressed with v-Src kinase in HEK293 cells to induce current suppression and decreased kinetics of inactivation {⁴¹}. Likewise, patch pipette dialysis of c-Src^pp60 plus MgATP induces similar biophysical changes in the outward whole-cell current of OB neurons, that is carried largely by Kv1.3 {¹²}. Although Src kinase is ubiquitously expressed throughout the CNS, it was important to confirm the expression of Src in the OB in addition to its capacity for modulation of Kv1.3. Ten or forty μg of OB lysate was separated by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. Membranes were probed with a monoclonal antibody for viral Src (v-Src, 1:40-1:100) and Src expression was detected in the OB lysates at the expected molecular weight of 60 kDa (Fig. 2).

In HEK 293 cells, a direct protein-protein interaction has been observed between v-Src kinase and Kv1.5 and this protein-protein interaction causes current suppression of Kv1.5 {⁷}. Although Src kinase modulates channel properties of Kv1.3 by tyrosine phosphorylation, the channel fails to co-immunoprecipitate with Src kinase in HEK 293 cells {⁷}. We conjectured that perhaps adaptor protein modules might facilitate channel-kinase interactions in native neurons expressing Src and Kv1.3. We thus probed the SDS-PAGE separated OB lysates with antisera for Shc (H-108) and Grb10 (K-20). Fig. 2 demonstrates the observed protein bands of the predicted molecular weight (46/52/66 kDa and 60 kDa, respectively) for these two adaptor proteins. Shc adaptor protein is not expressed in brain regions, however, a neuronal-specific member of the Shc family, n-Shc, is highly expressed in the brain and the OB, as shown by in situ hybridization{²⁹}. Since H-108 antibody detects all Shc isoforms as well as n-Shc, it is highly likely that we are detecting predominantly the n-Shc family member in the OB.

v-Src kinase phosphorylation of Kv1.3 is altered by adaptor proteins, Grb10 and n-Shc

Deletion of the c-Src activation loop produces the viral form of Src, v-Src, which has unlimited, constitutive activity in the absence of other cellular signaling {⁴⁶}. The rest of the experiments of this study were performed by transient co-transfection of v-Src plus Kv1.3 in an heterologous expression system (HEK 293 cells). This model facilitated the understanding of the potential mechanisms by which Src kinase could exert its effects on Kv1.3 in native OB neurons by allowing the experimenter to control the expression of putative protein-protein interacting partners and by having the ability to activate the kinase without induction of the endogenous upstream signaling cascade. Additionally, human embryonic kidney (HEK 293) cells are known to express high levels of cloned proteins {⁴⁷}, which facilitates good protein yields for immunoprecipitation. Untransfected HEK 293 cells also show no C-type inactivating K currents in response to voltage stimulation {⁴¹}.

HEK 293 cells were co-transfected with cDNA for Kv1.3 + v-Src (see Experimental Procedures) plus or minus cDNA coding n-Shc or Grb10 adaptor proteins. Cells were given approximately 40 hours to express proteins prior to cell harvest by lysis. Kv1.3 transfected cells always served as the control and total cDNA across various transfection conditions was balanced by non-coding pcDNA₃ vector. Precleared cell lysate was incubated overnight with the Kv1.3 directed antibody, α-AU13. Collected and washed immunoprecipitates (IPs) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with a phosphotyrosine-specific antibody (see Experimental Procedures; Upstate 4G10). A complementary approach yielding similar results was also used, in which phosphorylated proteins were precipitated from lysates with the phosphotyrosine-specific antibody (4G10), and separated proteins were probed for Kv1.3 (data not shown). Kv1.3 plus v-Src co-transfected HEK 293 cells demonstrated that v-Src kinase significantly increased tyrosine phosphorylation of Kv1.3 14-fold over that found in Kv1.3-alone transfected cells (n = 10) (Fig. 3). Kv1.3 exhibited low levels of basal tyrosine phosphorylation, but as also observed by Holmes et al. {⁴³}, its tyrosine phosphorylation increased dramatically when co-expressed with v-Src. The basal value of Kv1.3 tyrosine phosphorylation was assigned an arbitrary value of 1.0 (Fig. 3B). This value was used as a normalization factor for all other transfection conditions. The predicted molecular weight (M_r) of the Kv1.3 channel (58 kDa) shows a decreased electrophoretic mobility, i.e. upward band shift, on SDS-PAGE under protein tyrosine phosphorylation conditions {^{48, 49, 43}}. The bracketed region (Fig. 3A, upper panel) from 55-72 kDa spanning all Kv1.3 immunoreactivity was thus quantified in assessing Kv1.3 tyrosine phosphorylation in this and all subsequent experiments. The expression of Kv1.3 protein in each condition, for this, and all remaining experiments, was verified by separating 5 μg of cell lysate for each transfection condition and probing the lysates with α-Kv1.3 antibody (Fig. 3A, lower panel).

Both Grb10 and n-Shc adaptor proteins have been shown to be substrates of Src kinase {^{51, 51, 27}}, and each contain several signaling motifs for protein-protein interactions (Fig. 1). We tested the effect of each adaptor protein on v-Src-induced Kv1.3 phosphorylation using immunoprecipitation and Western analysis as described above. Addition of Grb10 cDNA to v-Src plus Kv1.3 co-transfected HEK 293 cells caused a significant decrease in Kv1.3 tyrosine phosphorylation, which approximated that of basal levels (Kv1.3 in the absence of v-Src kinase) (ANOVA, snk, α = 0.05) (Fig. 3B). Addition of n-Shc cDNA, however, slightly increased Kv1.3 tyrosine phosphorylation but not significantly greater than that found in Kv1.3 plus v-Src co-transfected cells (ANOVA, snk, α = 0.05) (Fig. 3B).

v-Src-induced modulation of Kv1.3 is altered by adaptor proteins, Grb10 and n-Shc

v-Src-induced current suppression of Kv1.3 {⁴¹} was first confirmed by comparing the biophysical properties of Kv1.3-transfected cells with those of Kv1.3-plus-v-Src-co-transfected cells. We then tested whether changes in v-Src-induced Kv1.3 tyrosine phosphorylation caused by co-expression with Grb10 or n-Shc also altered Kv1.3 physiology. For these patch-clamp experiments, HEK 293 cells were transfected with Kv1.3 alone or co-transfected with Kv1.3 plus v-Src, plus or minus an adaptor protein. Approximately 40 hours post-transfection, macroscopic outward currents were recorded in the cell-attached configuration. Cells were voltage-clamped at -90 mV and stepped to a depolarizing potential of +40 mV for 1 second to activate Kv1.3. Peak current amplitude was 1691.36 +/- 199.64 pA (n = 69) in the Kv1.3-only control transfection condition and 391.38 +/- 84.85 pA (n = 47) in the Kv1.3 plus v-Src condition, confirming a statistically significant 77% suppression of Kv1.3 current in the presence of the kinase (Student's t-test, α = 0.05) (Fig. 4A-B). This change in current magnitude appears to be independent of a shift in Kv1.3 voltage dependence. This is reflected in the lack of change of voltage at half-maximum activation (V_1/2) in the presence of v-Src as calculated from a Boltzman fit of the tail currents when cells were held at -90 mV and stepped to +20 mV in increments of 5 mV using a pulse duration of 50 ms (Table 1 and 2). v-Src significantly decreased the τ_Inact of Kv1.3 from 985 ms (n = 64) to 616 ms (n = 33), as shown in the examples of Figure 4C-D. v-Src also increased the τ_Deact of Kv1.3 from 34 ms (n = 63) to 43 ms (n = 40), although this did not represent a statistically significant difference (Student's t-test, α = 0.05) (Fig. 4E-F).

Upon co-transfection of Grb10 cDNA with Kv1.3 plus v-Src, Grb10 adaptor protein relieved v-Src-induced current suppression of Kv1.3 to return the peak current amplitude to 88% of the control level (Kv1.3-only transfection) (n = 19) (Fig. 4A, Table 1). Grb10 adaptor protein relieved the v-Src-induced decrease in τ_inact to values near those of the Kv1.3-only control condition, but Grb10 shifted τ_deact to values significantly lower than either the Kv1.3 or Kv1.3 plus v-Src transfection conditions (ANOVA, snk, α = 0.05) (Fig. 4C,E). In contrast, co-transfection of n-Shc cDNA only partially relieved v-Src-induced Kv1.3 current suppression (Fig. 4B, Table 2, n = 38). Kv1.3 current in the presence of v-Src and n-Shc was 59% of the Kv1.3-only control condition (Table 2). n-Shc also did not significantly relieve the v-Src-induced decrease in τ_inact but returned τ_deact to values similar to those of the Kv1.3-only transfection condition (ANOVA, snk, α = 0.05) (Fig. 4D,F).

Taken together, the data from our combined SDS-PAGE-immunoprecipitation and patch-clamp electrophysiological experiments indicate that the adaptor protein Grb10 inhibits a large proportion of the v-Src-induced tyrosine phosphorylation of Kv1.3 and that this decreased phosphorylation correlates with a decrease in Src kinase modulation of Kv1.3 current amplitude and kinetics. The adaptor molecule n-Shc does not significantly relieve v-Src-induced modulation of Kv1.3 current amplitude, the kinetics of inactivation, or the corresponding total tyrosine phosphorylation of the channel that is retained or increased in the presence of the adaptor protein. Although we do not know if different tyrosine residues in Kv1.3 are targeted by v-Src in the presence of n-Shc, clearly the structural differences between n-Shc and Grb10 could provide differential interaction with Kv1.3 to account for the differences in Kv1.3 physiology in the presence of Src kinase and n-Shc or Grb10. The fact that Grb10 inhibits most of v-Src-induced phosphorylation and functional modulation could suggest that Grb10 blocks access of v-Src kinase to Kv1.3 in the cell. Since both Grb10 and Kv1.3 contain proline-rich sequences specific for interaction with the SH3 domain of v-Src kinase, and the channel cannot tightly co-immunoprecipitate with Src at this domain {⁷}, Grb10 may have a stronger affinity for this domain and physically block the kinase access to the channel.

v-Src kinase disrupts Kv1.3 cumulative inactivation, an effect that is restored by Grb10 and accentuated by n-Shc

Kv1.3 ion channel inactivates slowly and remains in the inactivated state for up to 60 seconds {^{44, 52}}. When the interpulse interval is less than 60 seconds, Kv1.3 exhibits a cumulative inactivation upon repeated stimulation due to its long dwell time in the inactivated state. Kv1.3 cumulative inactivation has not been studied under a kinase-induced phosphorylated state. To test whether v-Src kinase affected Kv1.3 cumulative inactivation, cells were held at -90 mV and stepped to +40 mV 12 times for 1 second each at varying interpulse intervals. Peak current amplitudes were normalized to that of the first trace in each series of 12 traces and plotted against pulse number. Sample size for each interpulse interval varied slightly due to the technical difficulty of performing all needed recordings on a cell without losing the voltage-clamp. Kv1.3 showed the expected cumulative inactivation when expressed alone: no inactivation at 60 second interpulse intervals, slight cumulative inactivation was initiated at 30 second interpulse intervals, and marked cumulative inactivation was apparent at 10, 2, 1, and 0.5 s interpulse intervals (n = 22-26) (Fig. 5A). With an interpulse interval permitting only a 2 second recovery from inactivation, as much as 50% of the Kv1.3 channels were caught in the inactivated state. In the condition of v-Src co-transfection, Kv1.3 cumulative inactivation was not initiated until the interpulse interval was shortened to 2 seconds. A maximum of 20-25% of Kv1.3 channels were caught in the inactivated state, even when recovery from inactivation was only 500 milliseconds, compared with Kv1.3 alone transfections in which typically 75% of channels remain inactivated with such short recovery intervals (n = 19-23) (Fig. 5B). The severe decrease of Kv1.3 cumulative inactivation in the presence of the kinase was not due to overall kinase-induced current suppression because the current amplitudes were normalized to that of the first trace in each series and thus percent decreases could be calculated, if present. These data indicate that v-Src kinase could contribute a permissive function in the excitability of cells by increasing the number of Kv1.3 channels available for activation during rapid repetitive stimulation. The Src-induced disruption of Kv1.3 cumulative inactivation would be consistent with either Kv1.3 failing to enter the inactivated state or Kv1.3 exiting the inactivated state more quickly in the presence of the kinase. These alternatives could be best deciphered at the unitary current level in future studies.

Co-transfection of v-Src plus Kv1.3 cDNAs in the presence of either Grb10 or n-Shc adaptor proteins differentially affected the disruption of cumulative inactivation of Kv1.3 by v-Src kinase (Fig. 5C-D). Addition of n-Shc adaptor protein failed to restore cumulative inactivation of Kv1.3 in Kv1.3 plus v-Src co-transfected cells (Fig. 5D). When recovery from inactivation was shortened to only 500 milliseconds, nearly 90% of Kv1.3 channels could still be available for reactivation upon voltage stimulation. Addition of Grb10 adaptor protein, however, re-established cumulative inactivation properties of Kv1.3 in the presence of v-Src kinase (Fig. 5C).

The effects of Grb10 and n-Shc on the phosphorylation and modulation of Kv1.3 by v-Src are phosphorylation-dependent

To determine the importance of v-Src kinase activity and to eliminate the possibility that this kinase exerts its effects on Kv1.3 only through its own non-catalytic interaction domains, we substituted a mutant Src kinase for v-Src in our transfection scheme described above. Mutation of arginine 385 of Src to alanine produces a severely impaired Src kinase (R385A Src) {⁴⁰}. To confirm that the mutant Src kinase is in fact significantly impaired, we tested its ability to phosphorylate Kv1.3, and we show that this mutant only phosphorylates Kv1.3 to 4-fold above its basal level as opposed to the 14-fold increase by wild-type v-Src (n = 5) (Fig. 6B). The outward current of Kv1.3 in the presence of R385A Src kinase is 76% that of the controls, showing little suppression (n = 19) (Fig. 6A, Tables 1 and 2). The R385A Src kinase does not change the inactivation, deactivation, or cumulative inactivation of Kv1.3 in comparison to the control condition of Kv1.3 alone (n = 8) (Tables 1 and 2, Fig. 6A). When co-transfected with Kv1.3 plus R385A Src kinase, Grb10 decreases what little phosphorylation of Kv1.3 remains to less than that of the Kv1.3-only transfection condition (n = 3) (Fig. 6B). Physiologically, Kv1.3 peak current amplitude is increased 4-fold when R385A Src is substituted for Src kinase in the presence of Grb10, so there is no current suppression in this condition (n = 13) (Fig. 6A, Table 1). Grb10 fails to reverse the decreased τ_inact of the Kv1.3 plus v-Src co-transfection condition when R385A Src kinase is substituted for Src kinase (Table 1). Kv1.3 exhibits normal cumulative inactivation in the presence of R385A Src and Grb10 adaptor protein (n = 4-6) (Fig. 7B). These results demonstrate that the effects of Grb10 on v-Src-induced phosphorylation and subsequent modulation of Kv1.3 are dependent on the kinase activity of Src.

Transfection of n-Shc with Kv1.3 plus R385A Src results in no increase in phosphorylation over that of Kv1.3 + R385A Src (n = 3) compared with the 18-fold increase in channel tyrosine phosphorylation in the presence of the adaptor and live v-Src kinase (Fig. 6B). As with transfection of n-Shc with Kv1.3 + v-Src, substitution with R385A Src yielded an intermediate relief in v-Src-induced current suppression (Fig. 6A, Table 2) compared with control Kv1.3 alone conditions. Substitution of R385A Src kinase for v-Src also does not significantly reverse the v-Src-induced decrease in Kv1.3 τ_inact (Table 2). Kv1.3 exhibits normal cumulative inactivation in the presence of R385A Src and n-Shc adaptor protein (n = 2-3) (Fig. 7C). These results show that the lack of n-Shc effect on v-Src-induced Kv1.3 phosphorylation, Kv1.3 current suppression, decreased τ_inact, and cumulative inactivation are independent of an intact kinase domain of Src, whereas the n-Shc reversal effect on v-Src-induced 10 mV shift in V_1/2 and increase slope of voltage dependence (κ) is dependent on Src kinase activity. Taken together, the data from experiments using R385A Src suggest that Grb10 and n-Shc adaptor proteins each differently regulate the properties of Kv1.3 channel in the presence of v-Src kinase, and do so by a mechanism that requires Src kinase activity.

The CH domain is responsible for n-Shc regulation of v-Src-induced phosphorylation and current suppression of Kv1.3

Members of the Shc family of adaptor proteins contain highly homologous SH2 and PTB domains, responsible for recognizing specific sequences of other proteins containing phosphotyrosines, and CH domains that are not highly conserved and contain different varieties of proline-rich regions and tyrosine phosphorylation-specific sequences{²⁹}. Within the CH domain, the Shc protein is known to be phosphorylated on three specific tyrosine residues, tyr²³⁹ and tyr²⁴⁰, and tyr³¹⁷ {³⁵, 36, 51, 53, ⁵⁴} that correspond to tyr²²⁰, tyr²²¹, and tyr³⁰⁴ of n-Shc, respectively {²⁹}. Sck is a member of the Shc family, which is expressed predominantly in the peripheral nervous system, and differs from n-Shc in the structure of its CH domain. Sck is also NOT a substrate for Src kinase{²⁹}. Sck none the less contains homologous tyrosines in its CH domain. Dr. Takeshi Nakamura (Sumitomo Electric Industries) generously provided our laboratory with cDNA constructs encoding n-Shc, Sck, and a n-Shc mutant in which these three key tyrosines were conservatively mutated to phenylalanine (Y220F, Y221F, and Y304F). The cDNA construct in which all three tyrosine residues in the CH domain were collectively altered from tyrosine to phenylalanine as a cassette, we have termed “n-Shc triple tyrosine mutant.” We used this n-Shc triple tyrosine mutant and the Sck member of the Shc family to determine the contribution of the CH domain toward the n-Shc effect on v-Src-induced changes in Kv1.3 function and tyrosine phosphorylation. n-Shc triple tyrosine mutant lacks the tyrosine residues that would normally be phosphorylated by Src kinase to provide phosphotyrosine residues for interaction with SH2-containing proteins, whereas Sck contains the tyrosine residues, but these tyrosines are not normally substrates for Src kinase. We performed transfection of HEK 293 cells similar to that described for n-Shc above, substituting either Sck or the n-Shc triple tyrosine mutant for n-Shc.

When co-transfected with Kv1.3 and v-Src, Sck adaptor protein maintained an increased Kv1.3 phosphorylation by v-Src (n = 4) (Fig. 6B). Similar to n-Shc, Sck partially relieved v-Src-induced Kv1.3 current suppression to 56% of the control level (n = 21) (Fig. 6A, Table 2). Unlike n-Shc, however, Sck reversed the decreased τ_inact found in Kv1.3 plus v-Src co-transfected cells to an inactivation rate approaching Kv1.3 alone (Table 2). The increased τ_deact induced by v-Src kinase was decreased close to Kv1.3 control τ_deact when co-transfected with either n-Shc or Sck. Lastly, Sck, but not n-Shc, restored the cumulative inactivation of Kv1.3 in Kv1.3 plus v-Src co-transfected conditions (n = 10-11) (Fig. 7D). Results from these Sck experiments suggest that regions of the n-Shc CH domain that differ from those of the Sck CH domain are responsible for reversing the decreased τ_inact, the increased slope (κ) of voltage dependence, the right-shifted voltage at half-activation (V_1/2) and reestablishing the cumulative inactivation that becomes disrupted in v-Src + Kv1.3 co-transfected cells. The common inability to relieve current suppression and common ability to increase τ_deact back toward Kv1.3 control values compared with Kv1.3 + v-Src transfected conditions suggests that these properties are governed by CH domain residues in common across n-Shc and Sck.

When n-Shc triple tyrosine mutant was substituted for n-Shc in the transfection scheme of Kv1.3 plus v-Src kinase plus adaptor protein, phosphorylation of the ion channel was 8-fold over that of the control, slightly less than that of Kv1.3 plus v-Src kinase (n = 5) (Fig. 6B). Similar to that of wild-type n-Shc, the n-Shc triple tyrosine mutant relieved current suppression to 45% of that of the control (n = 7) (Fig. 6A, Table 2). In a manner similar to that of n-Shc, the n-Shc triple tyrosine mutant had no effect on τ_inact of Kv1.3 plus v-Src co-transfected cells but further decreased τ_deact to rates less than that of Kv1.3 alone. Similar to n-Shc, but not Sck, the n-Shc triple tyrosine mutant caused a 10 mV right shift in V_1/2 compared with Kv1.3 plus v-Src co-transfected cells lacking an adaptor molecule (Table 2). In contrast to n-Shc, but similar to Sck, the n-Shc triple tyrosine mutant restored cumulative inactivation of Kv1.3 to similar levels as that observed for Kv1.3 alone (n = 2) (Fig. 7E). The data from experiments performed with the n-Shc triple tyrosine mutant suggest that tyr ²²⁰, tyr²²¹, tyr³⁰⁴ are not important for increasing Kv1.3 phosphorylation by v-Src and are apparently also not necessary for the partial recovery of Src-induced Kv1.3 current suppression observed in the presence of n-Shc. Tyr²²⁰, tyr²²¹, and/or tyr³⁰⁴ in n-Shc, however, must participate in the loss of cumulative inactivation of Kv1.3 as disrupted by v-Src kinase, because removal of these residues (v-Src triple tyrosine mutant) or their capacity to be phosphorylated by Src (Skc) reverses Src disruption of Kv1.3 cumulative inactivation.

Substituting two proteins very similar to n-Shc with differences only in their CH domains has provided results suggesting that other residues than tyr²²⁰, tyr²²¹, and tyr³⁰⁴ of the n-Shc CH domain that are similar in the Sck CH domain are required for retaining or increasing v-Src-induced phosphorylation of Kv1.3. Differing regions of the CH domain, excluding the three key tyrosines, are important for reversing the decreased τ_inact observed in the Kv1.3 plus v-Src co-transfected condition. n-Shc, Sck, and the triple n-Shc mutant all show an intermediate, non-significant relief of v-Src-induced-Kv1.3 current suppression and all return τ_deact to rates less than that of the Kv1.3-only controls. Therefore, the ability for n-Shc to modulate current magnitude and τ_deact is likely to lie within its SH2 and/or PTB domains and not with residues or signaling structures contained in its CH domain.