The membrane protein MiRP3 regulates Kv4.2 channels in a KChIP-dependent manner

Abstract

MiRP3, the single-span membrane protein encoded by KCNE4, is localized by immunofluorescence microscopy to the transverse tubules of murine cardiac myocytes. MiRP3 is found to co-localize with Kv4.2 subunits that contribute to cardiac transient outward potassium currents (I(to)). Whole-cell, voltage-clamp recordings of human MiRP3 and Kv4.2 expressed in a clonal cell line (tsA201) reveal MiRP3 to modulate Kv4.2 current activation, inactivation and recovery from inactivation. MiRP3 shifts the half-maximal voltage for activation (V(1/2)) approximately 20 mV and slows time to peak approximately 100%. In addition, MiRP3 slows inactivation approximately 100%, speeds recovery from inactivation approximately 30%, and enhances restored currents so they 'overshoot' baseline levels. The cytoplasmic accessory subunit KChIP2 also assembles with Kv4.2 in tsA201 cells to increase peak current, shift V(1/2) approximately 5 mV, slow time to peak approximately 10%, slow inactivation approximately 100%, and speed recovery from inactivation approximately 250% without overshoot. Simultaneous expression of all three subunits yields a biophysical profile unlike either accessory subunit alone, abolishes MiRP3-induced overshoot, and allows biochemical isolation of the ternary complex. Thus, regional heterogeneity in cardiac expression of MiRP3, Kv4.2 and KChIP2 in health and disease may establish the local attributes and magnitude of cardiac I(to).

Figure 1. Spatial co-localization of MiRP3 and Kv4.2 within rat heart

Confocal images of rat left ventricle probed with antibodies to MiRP3 (A) and Kv4 (B). The T tubules appear yellow in the merged image (C). A differential interference contrast image (DIC, D) is provided for reference. Images E and F show similarly obtained confocal images when antibodies to MiRP3 and Kv4.2, respectively, were preincubated with blocking peptide; the blue colour reflects DAPI staining of nuclei. The white scale bar in B represents 20 μm.

Figure 2. MiRP3 modulates Kv4.2 activity

A, representative macroscopic whole-cell recordings made from tsA201 cells transfected with hKv4.2 ± hMiRP3, in response to a family of depolarizing pulses. Panel to the right shows Boltzmann fits to mean conductance values (± s.e.m., n = 13 and 10, respectively). B, sample traces for +40 mV pulses delivered after a 2.5 s prepulse. Graph shows Boltzmann fits to residual currents (relative to the first +40 mV pulse) as a function of the prepulse. C, representative currents from a two-pulse protocol with a variable interpulse interval at –100 mV. Currents were scaled so that the peak of the first pulse was 100%. Single-exponential fits to recovery from the two families shown yielded τ values of 80.5 ms and 62.3 ms in the absence and presence of MiRP3, with plateaus overshooting the first paired pulse by 0.1% and 10.9%, respectively. Fits to the mean per cent of recovery (± s.e.m.) are plotted as a function of the interpulse interval.

Figure 3. KChIP2 interferes with MiRP3 modulation of Kv4.2 kinetics

A–C, representative traces and fits to data (n = 16–21) obtained as described for Fig. 2, from tsA201 cells transfected with hKChIP2.1/hKv4.2 cDNA (in pIRES) and either empty vector or hMiRP3.

Figure 4. Stable biochemical interaction between MiRP3 and Kv4.2

COS-7 cells transfected with MiRP3 (a), MiRP3 and Kv4.2 (b), MiRP3 and Kv4.2–1d4 (c), or Kv4.2–1d4 alone (d), were subjected to lysis and immunopurification (IP) with anti-1d4 antibodies. An additional treatment group (e) consisted of lysate from cells expressing MiRP3 mixed with lysate from cells expressing Kv4.2–1d4, at 4°C for 16 h prior to immunopurification. Blots were probed with antibodies to Kv4.2 (70 kDa) and MiRP3 (21–25 kDa).

Figure 5. Ternary interactions between MiRP3, Kv4.2 and KChIP2

A, MiRP3 was immunopurified from lysates of tsA201 cells transfected with MiRP3 (a), MiRP3, Kv4.2 and KChIP2 (b), MiRP3 and KChIP2 (c), or Kv4.2 and KChIP2 (d). B, KChIP2 was immunoprecipitated from lysates of tsA201 cells transfected with KChIP2 (a), MiRP3 and KChIP2 (b), MiRP3, Kv4.2 and KChIP2 (c), Kv4.2 and KChIP2 (d), or MiRP3 and Kv4.2 (e). Images of blots span roughly 65–80 kDa for Kv4.2, 24–31 kDa for KChIP, and 20–28 kDa for MiRP3. Faint bands at 25–26 kDa in all five lanes of the IP blotted for MiRP3 are presumed to be crossreactivity of secondary antibodies to immunoprecipitated KChIP2 antibody.

Figure 6. Interactions between filamin A, MiRP3 and Kv4.2

A, COS-7 cells were transfected with fil–1d4 (a), MiRP3 and Kv4.2 (b), MiRP3, fil–1d4 and Kv4.2 (c), MiRP3 and fil–1d4 (d), or fil–1d4 and Kv4.2 (e). Cells were lysed and immunopurified (IP) with anti-1d4 antibodies. Note that low quantities of co-purified Kv4.2 required high-sensitivity scanning to detect signal. B, co-purification of MiRP3 with Kv4.2 in filamin-replete (A7) and filamin-deplete (M2) cells. Western blots show lysates and epitope-purified fractions from cells transfected with MiRP3 (a), Kv4.2–1d4 (b), or MiRP3 and Kv4.2–1d4 (c).