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Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the channel

Open AccessPublished:March 02, 2020DOI:https://doi.org/10.1074/jbc.RA119.011302
      The voltage-gated potassium channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1–S2 linker of the Kv1.5 channel. The low osmolarity–induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5. Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5. Our results further showed that the extracellular S1–S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1–S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1–S2 linker and made it no longer susceptible to proteinase K–mediated cleavage. In summary, the findings of our study indicate that the S1–S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this channel. By targeting the S1–S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic channel inhibition and results in an increase in IKv1.5.

      Introduction

      The voltage-gated potassium channel Kv1.5 mediates the ultra-rapid delayed rectifier potassium current (IKur)
      The abbreviations used are: IKur
      ultra-rapidly activating delayed rectifier potassium current
      PK
      proteinase K
      IKv1.5
      Kv1.5 current
      Kv1.5-HEK
      Kv1.5-expressing HEK cells
      LO
      low extracellular osmolarity
      MEM
      minimum essential medium
      CTL
      control
      Kv
      voltage-gated potassium
      V1/2
      voltage of half-maximal activation.
      in atrial myocytes (
      • Fedida D.
      • Wible B.
      • Wang Z.
      • Fermini B.
      • Faust F.
      • Nattel S.
      • Brown A.M.
      Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current.
      ), which is critical for atrial repolarization (
      • Wang Z.
      • Fermini B.
      • Nattel S.
      Sustained depolarization-induced outward current in human atrial myocytes: Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents.
      ). Kv1.5 also contributes to the regulation of vascular smooth muscle tone (
      • Archer S.L.
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      • Schremmer B.
      • Mercier J.C.
      • El Yaagoubi A.
      • Nguyen-Huu L.
      • Reeve H.L.
      • Hampl V.
      Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes.
      ,
      • Ohanyan V.
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      • Bardakjian R.
      • Kolz C.
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      • Kmetz J.
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      • Luli J.
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      • Khan N.
      • Hou H.
      • Kuppusamy P.
      • Graham J.
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      • et al.
      Requisite role of Kv1.5 channels in coronary metabolic dilation.
      ). The Kv1.5 channel is composed of four α-subunits; each subunit contains six transmembrane segments and cytoplasmic N and C termini. The extracellularly localized S1–S2 linker of Kv1.5 is unusually long, containing 54 amino acid residues compared with 5–44 amino acid residues for most other Kv channels (UniProt: P22460). We have previously demonstrated that extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1–S2 linker, separating the channel into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus) (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ). The N-fragments of Kv1.5 were found to interact with the rest of the channel to accomplish specific tasks related to channel function (
      • Lamothe S.M.
      • Hogan-Cann A.E.
      • Li W.
      • Guo J.
      • Yang T.
      • Tschirhart J.N.
      • Zhang S.
      The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.
      ).
      Dysfunction of IKur has been linked to atrial fibrillation, an arrhythmia prevalent in the elderly and patients with chronic heart failure (
      • Yang T.
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      Novel KCNA5 mutation implicates tyrosine kinase signaling in human atrial fibrillation.
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      Genetic variation in KCNA5: Impact on the atrial-specific potassium current IKur in patients with lone atrial fibrillation.
      ,
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      • Selby J.V.
      • Singer D.E.
      Prevalence of diagnosed atrial fibrillation in adults: National implications for rhythm management and stroke prevention. The AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) Study.
      ). Atrial fibrillation also develops from pathological conditions such as heart failure, hypertension, and dilated cardiomyopathy, conditions which involve myocardial stretch (
      • Anter E.
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      • Callans D.J.
      Atrial fibrillation and heart failure: Treatment considerations for a dual epidemic.
      ,
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      • Shantsila A.
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      Atrial fibrillation and hypertension.
      ,
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      Meta-analysis of atrial fibrillation in patients with various cardiomyopathies.
      ).
      Cardiomyocytes are exposed to mechanical forces, including stretch, compression, and shear. Alterations of mechanical forces may modify cardiomyocyte electrical, mechanical, metabolic, and structural properties. In this regard, mechano-gated ion channels have been identified as cardiac mechanoreceptors solely activated by a mechanical stimulus. They convert mechanical stimuli into electrical and biochemical signals, regulating various cellular processes in response to extracellular mechanical forces (
      • Peyronnet R.
      • Nerbonne J.M.
      • Kohl P.
      Cardiac mechano-gated ion channels and arrhythmias.
      ). In addition, some ion channels that are activated by nonmechanical stimuli can be mechanically modulated, leading to altered channel activity (
      • Peyronnet R.
      • Nerbonne J.M.
      • Kohl P.
      Cardiac mechano-gated ion channels and arrhythmias.
      ). In this regard, the voltage-gated Kv1.5 channel has been shown to be regulated by mechanical stretch. Guo et al. (
      • Guo W.
      • Kamiya K.
      • Kada K.
      • Kodama I.
      • Toyama J.
      Regulation of cardiac Kv1.5 K+ channel expression by cardiac fibroblasts and mechanical load in cultured newborn rat ventricular myocytes.
      ) reported that application of mechanical force by 48-h cyclic stretch at 0.5 Hz with 20% elongation in length increases voltage-gated Kv1.5 channel expression by 48% in cultured neonatal rat ventricular myocytes. Boycott et al. (
      • Boycott H.E.
      • Barbier C.S.
      • Eichel C.A.
      • Costa K.D.
      • Martins R.P.
      • Louault F.
      • Dilanian G.
      • Coulombe A.
      • Hatem S.N.
      • Balse E.
      Shear stress triggers insertion of voltage-gated potassium channels from intracellular compartments in atrial myocytes.
      ) found that the Kv1.5 channel is modulated by shear stress through mechanically induced redistribution of intracellular Kv1.5 into the sarcolemma of rat atrial myocytes. Nonetheless, the molecular mechanisms underlying the effects of mechanical force on Kv1.5 channel activity are not well-understood. In the present study, we demonstrate that mechanical stretch mediated by cell swelling and centrifugation increases Kv1.5 current (IKv1.5). We show that the S1–S2 linker may serve as a mechanosensor, mediating stretch-induced increase in IKv1.5 because of enhanced channel density in the plasma membrane through altered Src-interaction with the N terminus of the channel.

      Results

      Mechanical stretch by centrifugation increases IKv1.5

      During an experiment that involved centrifuging Kv1.5-expressing HEK (Kv1.5-HEK) cells, we unexpectedly discovered that centrifugation caused an increase in IKv1.5. We centrifuged Kv1.5-HEK cells at 70 × g for 5 min, and then suspended cells in minimum essential medium (MEM) for 20 min prior to whole-cell voltage clamp recordings. The centrifugation led to an increase in IKv1.5 compared with control (CTL, without centrifugation) (Fig. 1). Because relatively low-speed centrifugation allows Kv1.5-HEK cells to experience mechanical stretch from gravitational-force accelerations, this result suggests that channel activity is altered by mechanical force.
      Figure thumbnail gr1
      Figure 1Centrifugation increases IKv1.5. Kv1.5-HEK cells were centrifuged (CENTR) at 70 × g for 5 min. Cells were then re-suspended in normal culture medium for 20 min prior to IKv1.5 recordings. Kv1.5-HEK cells without centrifugation were used as control (CTL). Representative current traces along with the voltage protocol (top) and summarized I-V relationship (bottom) are shown. CTL, n = 29; CENTR, n = 38; **, p < 0.01 at 20 mV and above.

      LO treatment increases IKv1.5

      To further evaluate the effect of mechanical stretch on Kv1.5 activity, the osmotic potential of the cell culture medium was altered to induce cell swelling (
      • Montrose-Rafizadeh C.
      • Guggino W.B.
      Cell volume regulation in the nephron.
      ,
      • Chan H.C.
      • Nelson D.J.
      Chloride-dependent cation conductance activated during cellular shrinkage.
      ,
      • Volk T.
      • Frömter E.
      • Korbmacher C.
      Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.
      ). Specifically, standard MEM cell culture medium was mixed with purified distilled water at a 2:1 ratio, thus decreasing the osmolarity from 315 to 211 mOsm/liter (33% reduction). The degree of membrane stretch was examined using live cell imaging of Kv1.5-HEK cells cultured in isotonic (CTL) or hypotonic (LO) medium. Kv1.5-HEK cell size increased significantly after 30-min treatment with LO culture medium compared with control (Fig. 2A).
      Figure thumbnail gr2
      Figure 2LO medium treatment increases cell size and reversibly increases IKv1.5. A, culture of Kv1.5-HEK cells with LO medium for 30 min increased cell size (n = 13; **, p < 0.01). B, culture of Kv1.5-HEK cells with LO medium for 30 min increased IKv1.5. Representative current traces are depicted above the summarized I-V relationships (left) and activation curves (right). Activation curves were fitted to the Boltzmann function to determine V1/2 values and slope factors. CTL, n = 47; LO, n = 31; **, p < 0.01 at 20 mV and above for I-V curves; and tail currents at −30 mV following 50 mV depolarization for activation curves. C, LO-treatment mediated increase in IKv1.5 recovered (Recv) with time upon re-culture of cells in normal (isotonic) medium. Summarized I-V relationships at various time points were obtained from 7–27 cells. *, p < 0.05; **, p < 0.01 versus CTL at 20 mV and above; #, p < 0.05; ##, p < 0.01 versus LO at 20 mV and above.
      To examine the LO treatment on IKv1.5, Kv1.5-HEK cells were cultured in LO medium for various periods. The cells were then transferred to a perfusion chamber bathed with isotonic Tyrode solution, and IKv1.5 was recorded. LO-induced IKv1.5 increase occurred after 10 min (data not shown), reached maximum at 30 min (Fig. 2B), and did not develop further at 60 min (data not shown). The LO-induced increase in IKv1.5 at 30 min was not associated with a change in the activation curve (Fig. 2B, bottom right). The voltages of half-maximal activation (V1/2) were −7.6 ± 0.7 mV for control and −7.7 ± 0.7 mV for LO (p > 0.05). The slope factors were 6.1 ± 0.2 for control and 6.6 ± 0.2 for LO (p > 0.05).
      To examine whether LO increased IKv1.5 can recover upon re-culture in normal (isotonic) medium, we cultured multiple plates of Kv1.5-HEK cells in LO medium for 30 min. The culture media were then changed to normal (isotonic) medium. After various periods of culture, cells were collected and IKv1.5 was recorded. LO-induced IKv1.5 increase was reversible; The increased IKv1.5 was significantly recovered after 2 h and returned to control level after 4 h of re-culturing cells in normal medium (Fig. 2C).
      To determine whether the mechanical stretch-induced increase in IKv1.5 also occurs in native cardiac myocytes, we studied Kv1.5 channels expressed in neonatal rat ventricular myocytes. The endogenous Kv1.5 current in cardiac myocytes is small and complicated by the coexistence of other K+ channels. To overcome this issue, we transfected neonatal rat ventricular myocytes cultured on glass coverslips with human Kv1.5 plasmid. Twenty-four h after transfection, we treated the myocytes with LO media for 30 min. The cells were then transferred to the recording chamber and currents were recorded in isotonic Tyrode solution. As shown in Fig. 3, LO treatment for 30 min also significantly increased IKv1.5 in neonatal rat ventricular myocytes.
      Figure thumbnail gr3
      Figure 3LO medium treatment increases IKv1.5 in neonatal rat ventricular myocytes transfected with Kv1.5. Representative current traces are depicted above the summarized I-V relationship. CTL, n = 32; LO, n = 32. *, p < 0.05 at 20 mV and above.

      LO treatment–mediated current increase is specific to Kv1.5

      To determine whether LO treatment–mediated current increase observed in Kv1.5 also exists in other Kv channels, we treated HEK cells stably expressing Kv1.4, Kv4.3, Kv7.1+KCNE1, Kv10.1, or Kv11.1, respectively, with LO medium in culture for 30 min. After treatment, various currents were recorded in normal isotonic Tyrode solution. Although LO treatment slightly and nonsignificantly increased IKv4.3, it did not affect IKv1.4, IKv7.1+KCNE1, IKv10.1, or IKv11.1 (Fig. 4). Thus, LO treatment selectively increased IKv1.5.
      Figure thumbnail gr4
      Figure 4Mechanical stretch induced by LO medium culture selectively increases IKv1.5. Currents from Kv1.5, Kv1.4, Kv4.3, Kv7.1+KCNE1, Kv10.1, and Kv11.1 channels were recorded from HEK 293 cells stably expressing the respective channels in control cells and cells treated with LO medium for 30 min. Representative current traces are depicted above the summarized I-V relationships. For IKv1.5, CTL, n = 47; LO, n = 31 (same set of data shown in B). For IKv1.4, CTL, n = 43; LO, n = 46; for IKv4.3, CTL, n = 42; LO, n = 42; for IKv7.1+KCNE1, CTL, n = 15; LO, n = 12; for IKv10.1, CTL, n = 18, LO, n = 18; for IKv11.1, CTL, n = 23, LO, n = 19. **, p < 0.01 at 20 mV and above.

      The S1–S2 linker of Kv1.5 is involved in LO-induced current increase

      Compared with other Kv channels, the extracellular S1–S2 linker of the Kv1.5 channel is long and proline-rich, and uniquely contains an N-linked glycosylation site (Fig. 5A). To investigate whether the S1–S2 linker is involved in the mechanical stretch-induced increase in IKv1.5, we used the following strategies.
      Figure thumbnail gr5
      Figure 5The unique S1–S2 linker of Kv1.5 is involved in LO-mediated increase in IKv1.5. A, amino acid sequences of the S1–S2 linker of various Kv channels. Kv1.5 possesses an unusually long S1–S2 linker with 12 nonconserved proline residues (in magenta). The N-linked glycosylation site is shown in red. B, PK cleavage of the S1–S2 linker abolished LO-induced increase in IKv1.5. Schematic illustration of Kv1.5 PK cleavage (top) and representative current traces (middle) are depicted above the summarized I-V relationships (bottom). CTL, n = 14; LO, n = 21. C, mutating all 12 nonconserved prolines (P) to alanines (A) in the S1–S2 linker abolished LO-induced increase in IKv1.5. Schematic illustration of the Kv1.5–12PA mutant (top) as well as representative current traces (middle) are depicted above the summarized I-V relationships (bottom). CTL, n = 37; LO, n = 36. D, deletion of amino acid residues 282–300 in the S1–S2 linker abolished LO-induced increase in IKv1.5. Schematic illustration of Kv1.5-Δ282–300 mutant (top) as well as representative current traces (middle) are depicted above the summarized I-V relationships (bottom). CTL, n = 15, LO, n = 10. E, inhibition of glycosylation in the S1–S2 linker with tunicamycin (Tuni) treatment abolished the LO-induced increase in IKv1.5. Schematic illustration of WT Kv1.5 without glycosylation (top) as well as representative current traces (middle) are depicted above the summarized I-V relationships (bottom). CTL, n = 24; LO, n = 24.
      First, we examined whether an intact S1–S2 linker is required for LO-meditated IKv1.5 increase. We previously demonstrated that extracellularly applied PK precisely cleaves cell-surface Kv1.5 proteins at a single site in the S1–S2 linker, separating the 75-kDa channel protein into a 42-kDa N-fragment and a 33-kDa C-fragment (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ,
      • Lamothe S.M.
      • Hogan-Cann A.E.
      • Li W.
      • Guo J.
      • Yang T.
      • Tschirhart J.N.
      • Zhang S.
      The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.
      ). In the present study, we pretreated Kv1.5-HEK cells with PK (200 μg/ml, 20 min) and cultured cells in LO or normal (control) medium for 30 min. Cells were then transferred to the perfusion chamber to record IKv1.5 in isotonic Tyrode bath solution. PK pretreatment prevented the LO-mediated increase in IKv1.5, indicating that an intact S1–S2 linker is necessary for LO-mediated effect (Fig. 5B).
      Second, proline residues play important structural and functional roles for channel activity (
      • Wess J.
      • Nanavati S.
      • Vogel Z.
      • Maggio R.
      Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.
      ,
      • Hong S.
      • Ryu K.S.
      • Oh M.S.
      • Ji I.
      • Ji T.H.
      Roles of transmembrane prolines and proline-induced kinks of the lutropin/choriogonadotropin receptor.
      ,
      • Kaduk C.
      • Duclohier H.
      • Dathe M.
      • Wenschuh H.
      • Beyermann M.
      • Molle G.
      • Bienert M.
      Influence of proline position upon the ion channel activity of alamethicin.
      ,
      • Bett G.C.
      • Lis A.
      • Guo H.
      • Liu M.
      • Zhou Q.
      • Rasmusson R.L.
      Interaction of the S6 proline hinge with N-type and C-type inactivation in Kv1.4 channels.
      ). The Kv1.5 channel uniquely possesses 12 proline residues in the S1–S2 linker. To characterize the functional importance of these residues in the LO-mediated IKv1.5 increase, we created a mutant Kv1.5 channel, Kv1.5–12PA, in which all 12 nonconserved prolines (P) are replaced with alanines (A). LO treatment failed to affect Kv1.5–12PA current (Fig. 5C). Furthermore, we created another mutant Kv1.5 channel, Kv1.5-Δ282–300, in which amino acid residues from 282 to 300 in the S1–S2 linker were deleted. This led to removal of seven nonconserved prolines in the S1–S2 linker. Kv1.5-Δ282–300 current was also unaffected by LO treatment (Fig. 5D). Thus, an intact S1–S2 linker containing the nonconserved proline residues plays a role in the mechanical stretch-induced effect on Kv1.5 channel function.
      Third, Kv1.5 channels are glycosylated and N-linked glycosylation occurs at a site (Asn-299) located in the S1–S2 linker (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ). To investigate the role of glycosylation in LO-mediated increase in IKv1.5, we treated Kv1.5-HEK cells with tunicamycin (10 μg/ml) for 36 h, which led to a complete inhibition of N-glycosylation, as reported previously (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ,
      • Lamothe S.M.
      • Hogan-Cann A.E.
      • Li W.
      • Guo J.
      • Yang T.
      • Tschirhart J.N.
      • Zhang S.
      The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.
      ). LO treatment failed to affect IKv1.5 of channels treated with tunicamycin (Fig. 5E). Thus, glycosylation at a site in the S1–S2 linker plays an important role in LO-mediated Kv1.5 increase.

      The Kv1.5 channel N terminus is involved in the LO-mediated IKv1.5 increase

      The N terminus of Kv1.5 is an important target region for channel regulation. To investigate the involvement of the intracellular N terminus of the Kv1.5 channel in the LO-mediated effect, we deleted residues 1–209 of the N terminus, creating the ΔN209 mutant Kv1.5 channel, which was stably expressed in HEK293 (ΔN209 Kv1.5-HEK) cells. Truncation of the N terminus abolished LO-mediated current increase seen in WT Kv1.5 channels (Fig. 6, A and B). Thus, the N terminus of Kv1.5 plays a role in the LO-mediated increase in IKv1.5.
      Figure thumbnail gr6
      Figure 6The N terminus is involved in LO-mediated increase in IKv1.5. A, LO treatment for 30 min increased IKv1.5. Schematic illustration of WT Kv1.5 channel (top) and representative current traces (middle) are depicted above the summarized I-V relationships (bottom). CTL, n = 47; LO, n = 31; **, p < 0.01 at 20 mV and above (same set of data shown in B and for Kv1.5). B, N terminus truncation mutant ΔN209 abolished LO-induced increase in IKv1.5. Schematic illustration of Kv1.5-ΔN209 mutant (top) and representative current traces (middle) are depicted above the summarized I-V relationships (bottom). CTL, n = 13; LO, n = 11. C, LO treatment for 30 min had no effects on IKv1.4. Schematic illustration of WT Kv1.4 channel (top) and representative current traces (middle) are depicted above the summarized I-V relationships (bottom). CTL, n = 43; LO, n = 46 (same set of data shown in for Kv1.4); D, replacement of the N terminus of Kv1.5 with the N terminus of Kv1.4 prevented LO-induced increase in IKv1.5. Schematic illustration of Kv1.5–Kv1.4NT mutant (top) and representative current traces (bottom) are depicted above the summarized I-V relationships (bottom). CTL, n = 32; LO, n = 30. E, amino acid sequence alignment of the N termini of Kv1.5 (1–247, UniProt: P22460) and Kv1.4 (1–304, UniProt: P22459), with Kv1.5 SH3-binding motifs shown in blue.
      In contrast to Kv1.5, Kv1.4 was insensitive to LO treatment (Fig. 6C), and has a different N-terminal amino acid sequence (Fig. 6E). To validate the role of the Kv1.5 N terminus in LO-mediated increase in IKv1.5, we created a mutant Kv1.5 channel Kv1.5–Kv1.4NT, in which the N terminus of Kv1.5 is replaced with that of Kv1.4. The LO-mediated increase in IKv1.5 seen in WT Kv1.5 channels was not observed in the Kv1.5–Kv1.4NT mutant channel (Fig. 6, A and D). This result further confirms the importance of the Kv1.5 N terminus in LO-induced increase in IKv1.5.

      Disruption of Src-binding sites abolishes LO-induced increase in IKv1.5

      It has been shown that Src tyrosine kinase interacts with Kv1.5, leading to an inhibition of IKv1.5 (
      • Holmes T.C.
      • Fadool D.A.
      • Ren R.
      • Levitan I.B.
      Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
      ). The Src homology 3 (SH3) domain of Src binds to the proline-rich motif RPLPXXP in partner proteins (
      • Yu H.
      • Chen J.K.
      • Feng S.
      • Dalgarno D.C.
      • Brauer A.W.
      • Schreiber S.L.
      Structural basis for the binding of proline-rich peptides to SH3 domains.
      ). The N terminus between amino acid residues 65 and 82 of Kv1.5 contains two repeats of the SH3 domain–binding motif RPLPPLP (
      • Holmes T.C.
      • Fadool D.A.
      • Ren R.
      • Levitan I.B.
      Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
      ). However, Kv1.4 does not contain such a motif (Fig. 6E). To examine whether Src-mediated regulation of the Kv1.5 channel is involved in LO-induced increase in IKv1.5, we disrupted Src-channel interaction by deleting the Src-binding motifs from the Kv1.5 channel. Specifically, we deleted amino acids 64–82 of the Kv1.5 channel, creating Kv1.5-ΔPro (Kv1.5 with deletion of proline-rich Src-binding domains, Fig. 7A). Unlike WT Kv1.5 channels (Fig. 7B), Kv1.5-ΔPro mutant channels were unaffected by LO treatment (Fig. 7C).
      Figure thumbnail gr7
      Figure 7Removal of Src-binding sites abolishes LO-mediated increase in IKv1.5. A, amino acid sequences showing the two consensus SH3–binding motifs (RPLPPLP, shown in blue) in the N terminus of Kv1.5 as well as the mutant Kv1.5-ΔPro, in which amino acids 64–82 were removed. B, effects of LO treatment on WT IKv1.5. CTL, n = 36; LO, n = 27; **, p < 0.01 at 20 mV and above. C, removal of the Src-binding sites in Kv1.5 prevented LO-induced increase in IKv1.5. Representative current traces (top) are depicted above the summarized I-V relationships (bottom). CTL, n = 26; LO, n = 28.
      Cells such as HEK 293 cells and native cardiac myocytes express endogenous Src (
      • Holmes T.C.
      • Fadool D.A.
      • Ren R.
      • Levitan I.B.
      Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
      ,
      • Holmes T.C.
      • Fadool D.A.
      • Levitan I.B.
      Tyrosine phosphorylation of the Kv1.3 potassium channel.
      ,
      • Torsoni A.S.
      • Constancio S.S.
      • Nadruz Jr., W.
      • Hanks S.K.
      • Franchini K.G.
      Focal adhesion kinase is activated and mediates the early hypertrophic response to stretch in cardiac myocytes.
      ). We believe that Kv1.5 channels are under tonic inhibition by Src kinases. LO treatment may cause a relief of such tonic inhibition, leading to an increase in IKv1.5. To this end, we examined the effects of Src kinase inhibitor PP1 (
      • Hanke J.H.
      • Gardner J.P.
      • Dow R.L.
      • Changelian P.S.
      • Brissette W.H.
      • Weringer E.J.
      • Pollok B.A.
      • Connelly P.A.
      Discovery of a novel, potent, and Src family selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.
      ) and LO treatment on the expression and function of Kv1.5 channels. On Western blot analysis, Kv1.5 channels from whole-cell lysate display as a 68-kDa and a 75-kDa band (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ,
      • Lamothe S.M.
      • Hogan-Cann A.E.
      • Li W.
      • Guo J.
      • Yang T.
      • Tschirhart J.N.
      • Zhang S.
      The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.
      ). The former represents the core-glycosylated immature protein inside the cell, and the latter represents the fully glycosylated mature protein normally localized in the plasma membrane (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ). Our results revealed that neither LO treatment (30 min) nor PP1 treatment (10 μm, 1 h) affected the total amount of mature (75-kDa) channel expression in whole cell lysate (Fig. 8, A and B, left lanes). It has been reported that mechanical stretch leads to redistribution of Kv1.5 channels toward the plasma membrane (
      • Boycott H.E.
      • Barbier C.S.
      • Eichel C.A.
      • Costa K.D.
      • Martins R.P.
      • Louault F.
      • Dilanian G.
      • Coulombe A.
      • Hatem S.N.
      • Balse E.
      Shear stress triggers insertion of voltage-gated potassium channels from intracellular compartments in atrial myocytes.
      ). To address this possibility, we isolated cell surface protein using biotinylation (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ). LO treatment as well as PP1 treatment increased the density of cell-surface 75-kDa channels (Fig. 8, A and B, right lanes). Indeed, incubation of Kv1.5-HEK cells with Src inhibitor PP1 (10 μm) for 1 h led to an increase in IKv1.5, similar to the LO-mediated IKv1.5 increase (Fig. 8C). Furthermore, after pretreatment with Src inhibitor PP1, LO treatment no longer increased IKv1.5 (Fig. 8C). These results suggest that LO increases IKv1.5 by disrupting Src interaction with the channel, which leads to accumulation of Kv1.5 channels on the plasma membrane.
      Figure thumbnail gr8
      Figure 8Effects of LO-treatment and Src inhibitor PP1 on Kv1.5 expression and function. A, LO treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in LO-treated cells was normalized to that of control cells in the same gel and shown in the scatter plots (n = 5). B, PP1 treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in PP1-treated cells was normalized to that of control cells in the same gel and shown in the scatter plot (n = 5). For A and B, boxes represent interquartile ranges, horizontal lines represent medians, whiskers represent 5–95% ranges, and gray boxes represent means. **, p < 0.01 versus CTL. C, Src inhibitor PP1 treatment increased IKv1.5 and prevented LO-mediated increase in IKv1.5. Representative current traces (top) are depicted above the summarized I-V relationships (bottom). CTL, n = 32; PP1, n = 35; LO, n = 26; PP1+LO, n = 28. *, p < 0.05 at 20 mV and above, compared with control (CTL). There was no significant difference among PP1, LO, and PP1+LO groups.

      The Kv1.5 S1–S2 linker communicates with the N terminus

      Our data so far indicate that both the S1–S2 linker and the N terminus are involved in the mechanical stretch-mediated increase in IKv1.5. We propose that mechanical stretch increases membrane channel density and IKv1.5 through a conformational change in the N terminus mediated via the S1–S2 linker, which releases Src-channel interaction. To test this notion, we examined the communication between the S1–S2 linker and N terminus.
      To investigate whether the extracellular S1–S2 linker conformation can be affected by a modified N terminus, we used the N-terminal truncation channel ΔN209 that is functional (Fig. 6B). Compared with WT channel proteins, Kv1.5 ΔN209 has a smaller molecular mass. Western blot analysis of ΔN209 displays a 50-kDa band, representing the mature channel protein, and a 45-kDa band, representing the immature protein. We have previously demonstrated that extracellularly applied PK (200 μg/ml, 20 min) cleaves the 75-kDa protein of WT channels at a site in the S1–S2 linker, generating an N-fragment and a C-fragment (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ,
      • Lamothe S.M.
      • Hogan-Cann A.E.
      • Li W.
      • Guo J.
      • Yang T.
      • Tschirhart J.N.
      • Zhang S.
      The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.
      ). Consistently, PK treatment selectively cleaved the cell-surface 75-kDa band (without affecting the intracellularly localized 68-kDa band), generating a 33-kDa band detected with a C-terminal antibody (Fig. 9A). However, despite the fact that the 50-kDa ΔN209 protein is also located in the cell-surface to generate current, PK treatment could not cleave it (Fig. 9B). Thus, shortening of the intracellular N terminus causes a conformational change in the extracellular S1–S2 linker of the channel, making the linker resistant to cleavage by PK (Fig. 9). These data provide direct evidence that the S1–S2 linker communicates with the N terminus in a conformational manner.
      Figure thumbnail gr9
      Figure 9The Kv1.5 S1–S2 linker communicates with the N terminus in a conformational manner. Truncation of N terminus altered the susceptibility of the S1–S2 linker to PK cleavage. WT Kv1.5 displays 75-kDa and 68-kDa bands on Western blot analysis. The 75-kDa band represents the mature, fully glycosylated channel protein in the plasma membrane, whereas the 68-kDa band represents the immature channel protein inside the cell. ΔN209 Kv1.5 also presents as two bands; the 50-kDa band represents mature protein in the plasma membrane, the 43-kDa band represents immature protein inside the cell. Although PK completely cleaved the mature (cell surface) channel proteins of WT Kv1.5, it failed to cleave the mature (cell surface) channel proteins of ΔN209 Kv1.5 (n = 6).

      Discussion

      Although ion channels can be activated by a single primary stimulus, some are polymodal and can be activated by multiple types of stimuli such as voltage and temperature (
      • Yang F.
      • Zheng J.
      High temperature sensitivity is intrinsic to voltage-gated potassium channels.
      ). Because Kv1.5-expressing atrial myocytes and vascular smooth muscle cells are exposed to mechanical forces that may change in various conditions, investigating the role of mechanical force on Kv1.5 is an important step toward understanding the regulation and role of this channel in pathophysiological conditions.
      Our results revealed that IKv1.5 was increased by centrifugation (Fig. 1) and LO treatment (Figure 2, Figure 3), and LO-mediated current increase was specific to the Kv1.5 channel among Kv channels tested in the present study (Fig. 4). Centrifugation exerts gravitational force on cells causing membrane stretch against the interior of the centrifugation tube, leading to an increase in IKv1.5. Membrane stretch induced by hypotonic solution is an established strategy to study the effect of mechanical force on ion channels (
      • Gomis A.
      • Soriano S.
      • Belmonte C.
      • Viana F.
      Hypoosmotic- and pressure-induced membrane stretch activate TRPC5 channels.
      ,
      • Linz P.
      • Veelken R.
      Serotonin 5-HT(3) receptors on mechanosensitive neurons with cardiac afferents.
      ,
      • Schmidt D.
      • del Mármol J.
      • MacKinnon R.
      Mechanistic basis for low threshold mechanosensitivity in voltage-dependent K+ channels.
      ). In our study, Kv1.5-HEK cells were cultured with LO media for 30 min, then currents were recorded in normal, isotonic Tyrode solution. This LO treatment increased IKv1.5 (Fig. 2). However, LO treatment during whole-cell voltage clamp recordings did not affect IKv1.5 (data not shown). These observations are consistent with our finding that intracellular Src signaling (Figure 6, Figure 7) and associated protein trafficking (Fig. 8) are involved in LO-mediated IKv1.5 increase. The disturbance of the intercellular environment by the whole-cell patch clamp configuration (
      • Zhang S.
      • Hiraoka M.
      • Hirano Y.
      Effects of α1-adrenergic stimulation on L-type Ca2+ current in rat ventricular myocytes.
      ) likely prevented the LO treatment–mediated IKv1.5 increase.
      Detecting mechanical forces exerted on cells requires a sensor. Transmembrane cytoskeletal proteins are able to detect changes in membrane lipid structure and are involved in transduction of mechanical force (
      • Fonseca P.M.
      • Inoue R.Y.
      • Kobarg C.B.
      • Crosara-Alberto D.P.
      • Kobarg J.
      • Franchini K.G.
      Targeting to C-terminal myosin heavy chain may explain mechanotransduction involving focal adhesion kinase in cardiac myocytes.
      ,
      • Han B.
      • Bai X.H.
      • Lodyga M.
      • Xu J.
      • Yang B.B.
      • Keshavjee S.
      • Post M.
      • Liu M.
      Conversion of mechanical force into biochemical signaling.
      ,
      • Katsumi A.
      • Naoe T.
      • Matsushita T.
      • Kaibuchi K.
      • Schwartz M.A.
      Integrin activation and matrix binding mediate cellular responses to mechanical stretch.
      ). As well, proline (Pro) residues, which provide rigidity to the polypeptide chain, are proposed to play important structural and functional roles for channel activity (
      • Wess J.
      • Nanavati S.
      • Vogel Z.
      • Maggio R.
      Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.
      ,
      • Hong S.
      • Ryu K.S.
      • Oh M.S.
      • Ji I.
      • Ji T.H.
      Roles of transmembrane prolines and proline-induced kinks of the lutropin/choriogonadotropin receptor.
      ,
      • Kaduk C.
      • Duclohier H.
      • Dathe M.
      • Wenschuh H.
      • Beyermann M.
      • Molle G.
      • Bienert M.
      Influence of proline position upon the ion channel activity of alamethicin.
      ,
      • Bett G.C.
      • Lis A.
      • Guo H.
      • Liu M.
      • Zhou Q.
      • Rasmusson R.L.
      Interaction of the S6 proline hinge with N-type and C-type inactivation in Kv1.4 channels.
      ). This led to our investigation of the uniquely long and proline-rich extracellular S1–S2 linker of Kv1.5 channels. The LO-mediated increase in IKv1.5 was abolished by cleavage of the S1–S2 linker using PK treatment, replacement of 12 nonconserved prolines in the S1–S2 linker with alanines, or deletion of residues 282–300 from the S1–S2 linker (Fig. 5, B–D). Furthermore, the N-linked glycosylation is located in the middle (Asn-299) of the extracellular S1–S2 linker. Our results showed that inhibition of glycosylation abolished LO-mediated increase in IKv1.5 (Fig. 5E). Thus, the bulky, extracellular exposed glycan also plays a role in sensing mechanical forces. Overall, our results revealed a novel role of the extracellular S1–S2 linker as a sensor, capable of converting membrane stretch into biochemical signals that affect channel function. In line with this, the atypical structure of the Kv1.5 S1–S2 linker provides an explanation for how mechanical stretch is specific to the Kv1.5 channel among various Kv channels examined (Fig. 4).
      Our results also revealed that the N terminus is involved in the LO-mediated increase in IKv1.5 (Fig. 6). The current of the N-terminal truncation mutant, ΔN209, was unresponsive to LO treatment (Fig. 6B). The involvement of the Kv1.5 N terminus was further determined by creating a Kv1.5 mutant channel Kv1.5–Kv1.4NT, in which the N terminus of Kv1.5 was replaced by the N terminus of Kv1.4, a Kv channel that is unaffected by LO treatment (Fig. 6C). Kv1.5–Kv1.4NT channels were not affected by LO treatment (Fig. 6D). The N terminus of the Kv1.5 channel contains a proline-rich motif for binding of Src kinase, and the Src-channel interaction leads to a suppression of IKv1.5 (
      • Holmes T.C.
      • Fadool D.A.
      • Ren R.
      • Levitan I.B.
      Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
      ). Endogenous Src exists in HEK cells and cardiac myocytes (
      • Holmes T.C.
      • Fadool D.A.
      • Ren R.
      • Levitan I.B.
      Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
      ,
      • Holmes T.C.
      • Fadool D.A.
      • Levitan I.B.
      Tyrosine phosphorylation of the Kv1.3 potassium channel.
      ,
      • Torsoni A.S.
      • Constancio S.S.
      • Nadruz Jr., W.
      • Hanks S.K.
      • Franchini K.G.
      Focal adhesion kinase is activated and mediates the early hypertrophic response to stretch in cardiac myocytes.
      ), and association between Kv1.5 and native Src has been demonstrated in human myocardium tissue lysates (
      • Holmes T.C.
      • Fadool D.A.
      • Ren R.
      • Levitan I.B.
      Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.
      ). Our results revealed that inhibition of Src tyrosine kinase by PP1 increased IKv1.5 to an extent similar to LO treatment. Furthermore, after PP1 pretreatment, LO treatment no longer increased IKv1.5 (Fig. 8C). To confirm the role of Src tyrosine kinase binding in mechanical stretch, we created a Kv1.5 mutant channel, Kv1.5-ΔPro, by deleting the SH3 domain–binding motifs within the N terminus (Fig. 7). Removal of the Kv1.5 channel Src-binding sites led to an increase in IKv1.5 (note the current amplitude between Fig. 7, B and C), consistent with the notion that Kv1.5 channel activity is constitutively inhibited by endogenous Src kinases. Deletion of Src-binding sites abolished the LO-mediated current increase (Fig. 7C). These results suggest that LO treatment is likely to increase IKv1.5 by relieving the constitutive inhibition caused by endogenous Src tyrosine kinase.
      We then investigated how LO treatment affects the interaction of the N terminus with Src kinase. Our results revealed that the N terminus communicates with the S1–S2 linker. Although PK completely cleaved the mature protein of WT Kv1.5 channels, it could not cleave the mature protein of the N-terminal truncation mutant, Kv1.5 ΔN209 (Fig. 9). The S1–S2 linker of Kv1.5 is exposed extracellularly, whereas the N terminus is localized intracellularly. Thus, the resistance of Kv1.5 ΔN209 to PK cleavage provides direct evidence that shortening the intracellularly localized N terminus affects the conformation of the extracellularly localized S1–S2 linker, preventing cleavage by PK. Inversely, we believe that a change in the S1–S2 linker induced by mechanical forces may lead to conformational alteration of the N terminus. Such a change may cause Src dissociation and relief of tonic inhibition of the channel, leading to an increase in IKv1.5. It remains to be elucidated which and how extracellular matrix molecules are coupled to the S1–S2 linker of Kv1.5 channels to affect channel function. Nonetheless, our study raised the possibility that a mechanical stimulus can act on the extracellular S1–S2 linker that allosterically couples to an intracellular event (e.g. Src dissociation and relief of inhibition). Because Kv1.5 plays an important role in atrial repolarization (
      • Fedida D.
      • Wible B.
      • Wang Z.
      • Fermini B.
      • Faust F.
      • Nattel S.
      • Brown A.M.
      Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current.
      ,
      • Wang Z.
      • Fermini B.
      • Nattel S.
      Sustained depolarization-induced outward current in human atrial myocytes: Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents.
      ) and atrial stretch is associated with atrial fibrillation (
      • De Jong A.M.
      • Maass A.H.
      • Oberdorf-Maass S.U.
      • Van Veldhuisen D.J.
      • Van Gilst W.H.
      • Van Gelder I.C.
      Mechanisms of atrial structural changes caused by stretch occurring before and during early atrial fibrillation.
      ), the role of Kv1.5 in this association warrants further investigation.
      It is not well-understood how Src binding to the channel causes an inhibition of channel currents. Our results showed that the LO-induced increase in IKv1.5 was not associated with a change in the activation-voltage relationship of the channel (Fig. 2B). Boycott et al. (
      • Boycott H.E.
      • Barbier C.S.
      • Eichel C.A.
      • Costa K.D.
      • Martins R.P.
      • Louault F.
      • Dilanian G.
      • Coulombe A.
      • Hatem S.N.
      • Balse E.
      Shear stress triggers insertion of voltage-gated potassium channels from intracellular compartments in atrial myocytes.
      ) reported that shear stress triggers plasma membrane insertion of Kv1.5 channels from intracellular compartments in atrial myocytes. Consistently, our results showed that LO and PP1 treatment increased the cell surface Kv1.5 expression without affecting the total amount of Kv1.5 proteins from whole-cell lysate (Fig. 8, A and B). This result suggests that disruption of the endogenous Src-channel interaction enhances the longevity of Kv1.5 channels on the plasma membrane, leading to accumulated membrane expression and increased IKv1.5.
      In summary, by subjecting cells to centrifugation and hypotonic solution, our study revealed mechanical stretch as a novel pathway for Kv1.5 channel regulation. Mechanical stretch is detected by the S1–S2 linker of the Kv1.5 channel, which communicates with the N terminus, relieving Src-mediated tonic inhibition, leading to an increase in IKv1.5.

      Experimental procedures

      Molecular biology

      WT human Kv1.5 cDNA was provided by Dr. Michael Tamkun (Colorado State University, Fort Collins, CO). Human ether-a-go-go related gene (hERG) cDNA was provided by Dr. Gail Robertson (University of Wisconsin-Madison). Human ether-a-go-go gene (hEAG) was provided by Dr. Luis Pardo (Max-Planck Institute of Experimental Medicine, Göttingen, Germany). Human KCNQ1 and KCNE1 cDNAs were provided by Dr. Michael Sanguinetti (University of Utah, Salt Lake City, UT). Human Kv4.3 was provided by Gui-Rong Li (University of Hong Kong, Hong Kong, China). Kv1.4 cDNA was purchased from GenScript. Mutations in Kv1.5 channel, including Kv1.5-ΔN209 (deletion of amino acids 1–209), Kv1.5-Δ282–300 (deletion of amino acids 282–300 in S1–S2 linker), Kv1.5-ΔPro (deletion of amino acids 64–82 which contains Src-binding motifs), Kv1.5–12PA (mutating all 12 prolines to alanines in the S1–S2 linker), and Kv1.5–Kv1.4NT (the N terminus of Kv1.5 replaced by the N terminus of Kv1.4) were generated by PCR cloning of the corresponding constructs from WT Kv1.5 and Kv1.4 template and using BamH1 and EcoR1 restriction enzymes in the pcDNA3 vector. All sequencings were verified by GENEWIZ (South Plainfield, NJ). Human embryonic kidney (HEK) 293 cell lines stably expressing Kv1.5, Kv1.4, Kv4.3, KCNQ1 (Kv7.1) + KCNE1, hEAG (Kv10.1), hERG (Kv11.1), or each of the mutant Kv1.5 channels were created using Lipofectamine 2000 to transfect the respective plasmid into HEK 293 cells, followed by G418 for selection (1 mg/ml) and maintenance (0.4 mg/ml). Cells were cultured in MEM supplemented with 10% FBS, 1 mm sodium pyruvate, and 1% nonessential amino acids (Thermo Fisher Scientific).

      Neonatal rat ventricular myocyte isolation and culture

      Neonatal rat experiments were approved by the Queen's University Animal Care Committee. Ventricles of Sprague-Dawley rats of either sex at 1 day old were used to isolate ventricular myocytes using enzymatic dissociation (
      • Tschirhart J.N.
      • Li W.
      • Guo J.
      • Zhang S.
      Blockade of the human ether a-go-go related gene (hERG) potassium channel by fentanyl.
      ). Isolated ventricular myocytes were initially cultured in 10% FBS-containing DF medium for 45 min to allow fibroblasts to adhere to the bottom of the plate. Unadhered ventricular myocytes were then transferred to culture plates with glass coverslips and cultured overnight. Kv1.5 and GFP plasmids at a 4:1 ratio were transfected to ventricular myocytes using Lipofectamine 2000. Twenty-four h after transfection, GFP-positive cells were used to record IKv1.5 using the same voltage protocols and solutions as in HEK cells.

      Mechanical stretch induced by centrifugation and LO treatment

      To induce mechanical stretch, Kv1.5-HEK cells were detached from plates with trypsin, placed in tubes, and centrifuged at 70 × g for 5 min. The centrifuged cells were then resuspended and transferred for patch clamp recordings. Control cells were collected in the same way, but without centrifugation (detached with trypsin and placed in tubes). We also induced mechanical stretch in Kv1.5-HEK cells with LO medium culture for 30 min. LO media were created by diluting standard cell culture medium (MEM with 10% FBS) with purified water at a 2:1 ratio, decreasing the osmolarity of the medium from 315 to 211 mOsm/liter (33% reduction).

      Extracellular cleavage of cell surface proteins

      To cleave the extracellularly localized S1–S2 linker of Kv1.5 channels, the cells were treated with the serine protease PK (200 μg/ml in MEM) for 20 min at 37 °C. PBS containing 6 mm PMSF and 25 mm EDTA was then used to terminate the reaction. Biochemical and electrophysiological experiments were performed to detect the channel protein expression and current. Kv1.5-HEK cells treated with 250 μg/ml trypsin in PBS for 20 min at 37 °C were used as control.

      Western blot analysis

      Whole-cell protein lysates were used for Western blot analysis using the procedure described previously (
      • Hogan-Cann A.
      • Li W.
      • Guo J.
      • Yang T.
      • Zhang S.
      Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.
      ,
      • Lamothe S.M.
      • Hogan-Cann A.E.
      • Li W.
      • Guo J.
      • Yang T.
      • Tschirhart J.N.
      • Zhang S.
      The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.
      ). Cells were washed and collected with ice-cold PBS and centrifuged for 4 min at 100 × g. Cell pellets were then lysed in ice-cold lysis buffer containing 1 mm PMSF and 1% protease inhibitor mixture using sonification. Next, the cell lysates were centrifuged for 10 min at 10,000 × g, and supernatants containing proteins were collected. A protein assay kit (Bio-Rad) was used to determine protein concentrations. To create 0.3 μg/μl protein samples the appropriate amounts of double-distilled water and loading buffer containing 5% β-mercaptoethanol were added to the protein. Proteins samples of 15 μg were loaded and separated on 8% SDS-polyacrylamide gels and transferred onto PVDF membranes. To prevent nonspecific protein interactions, membranes were blocked with 5% nonfat skim milk and 0.1% Tween 20 in TBS for 1 h at room temperature. The blots were incubated with a C terminus–specific rabbit anti-Kv1.5 primary antibody (APC-004, Alomone) for 1 h at room temperature and then with goat anti-rabbit HRP-conjugated secondary antibody for 1 h. The blots were visualized with Fuji X-ray films (Fujifilm, Tokyo, Japan) using an enhanced chemiluminescence detection kit (GE Healthcare).

      Isolation of cell surface proteins

      Cell membrane proteins were isolated using biotinylation method with a Cell Surface Protein Isolation Kit (89881, Thermo Fisher Scientific). Specifically, cells at 90% confluence on 100-mm plates were treated with a membrane-impermeable thiol-cleavable amine-reactive biotinylation reagent, Sulfo-NHS-SS-biotin (250 μg/ml) for 30 min at 4 °C. Quenching solution was then added, and cells were lysed. After centrifugation at 10,000 × g for 2 min at 4 °C, biotin-labeled proteins were isolated using NeutrAvidin agarose columns and eluted with SDS-polyacrylamide sample buffer containing DTT. The isolated cell surface proteins were analyzed using Western blot analysis to detect Kv1.5 membrane expression. To ensure equal loadings, Na+/K+ ATPase expression was detected with a mouse anti-Na+/K+-ATPase α-1 antibody and a horse anti-mouse HRP-conjugated secondary antibody.

      Electrophysiological recordings

      All currents were recorded using whole-cell voltage clamp method. Cells collected were allowed to settle on the bottom of a 0.5 ml perfusion chamber in bath solution. The glass pipettes were pulled using thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL). The pipettes had inner diameters of ˜1.5 μm and resistances of 2 mΩ; when filled with solution. Series resistance (Rs) was compensated by 80%, and leak subtraction was not used. An Axopatch 200B amplifier and pCLAMP10 (Molecular Devices, San Jose, CA) were used for data acquisition and analysis. Data were sampled at 20 kHz and filtered at 5 kHz. The bath solution contained (in mm) 135 NaCl, 5 KCl, 10 HEPES, 10 glucose, 1 MgCl2, and 2 CaCl2 (pH 7.4 with NaOH). The pipette solution contained (in mm) 135 KCl, 5 EGTA, 5 MgATP, and 10 HEPES (pH 7.2 with KOH). For whole-cell voltage clamp recordings, currents were elicited from a holding potential of −80 mV by depolarizing steps to voltages between −70 and +70 mV in 10-mV increments. The membrane was then clamped to −50 mV (−30 mV for Kv1.5) prior to returning to the holding potential. Current amplitudes were normalized to the cell capacitances and expressed as current density (pA/pF). For current-voltage relationships of all channels, current amplitudes upon depolarizing steps were measured and plotted against voltages. For the Kv1.5 activation curve, peak tail currents during the −30 mV repolarizing step were measured, and plotted against the depolarizing voltages; the tail current-voltage relationships were fitted to the Boltzmann equation to obtain half-activation voltages (V1/2) and slope factors. Patch clamp experiments were performed at room temperature (22 ± 1 °C).

      Live cell microscopy

      WT Kv1.5-HEK cells were cultured on glass-bottom plates (35 mm) (World Precision Instruments, Sarasota, FL). Before and after culture with LO medium for 30 min at 37 °C, cell images were obtained using a 63× oil objective on a Zeiss Z.1 AxioObserver inverted microscope (Zeiss, Oberkochen, Germany). To quantify cell size, Zeiss AxioVision software was used.

      Reagents and antibodies

      A C terminus–specific rabbit anti-Kv1.5 antibody (APC-004) was purchased from Alomone Labs (Jerusalem, Israel). A mouse anti-Na+/K+-ATPase α-1 (sc-21712) primary antibody was purchased from Santa Cruz Biotechnology (Dallas, TX). Horse anti-mouse (7076) and goat anti-rabbit (7074) HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). MEM, FBS, trypsin, sodium pyruvate, minimal essential amino acids, Lipofectamine 2000, and Opti-MEM were purchased from Thermo Fisher Scientific. G418, PMSF, protease inhibitor mixture, β-mercaptoethanol, proteinase K, Triton X-100, BSA, and all chemicals/electrolytes used in the patch clamp experiments were obtained from Sigma-Aldrich. The BLUeye Prestained Protein Ladder (GeneDirex) was purchased from FroggaBio (Toronto, Ontario, Canada). An enhanced chemiluminescence detection kit was purchased from GE Healthcare. X-ray films were from Fujifilm (Tokyo, Japan).

      Statistical analysis

      All data are expressed as the mean ± S.E. For experiments with multiple groups, a two-way analysis of variance with Bonferroni post hoc test was used. For experiments between two groups, a two-tailed unpaired Student’s t test was used, except for Fig. 2A where a two-tailed paired Student’s t test was used. A p value ≤ 0.05 was considered statistically significant.

      Data availability

      All the data are in the manuscript.

      Author contributions

      A. O. M., T. W., W. L., and J. G. data curation; A. O. M., T. W., W. L., and J. G. formal analysis; A. O. M., T. W., and S. Z. investigation; A. O. M. and T. W. writing-original draft; A. O. M., T. W., W. L., J. G., and S. Z. writing-review and editing; S. Z. conceptualization; S. Z. supervision; S. Z. funding acquisition; S. Z. validation; S. Z. project administration.

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