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. 2008 Sep;45(3):336-46.
doi: 10.1016/j.yjmcc.2008.05.001. Epub 2008 May 12.

Post-transcriptional gene silencing of KChIP2 and Navbeta1 in neonatal rat cardiac myocytes reveals a functional association between Na and Ito currents

Isabelle Deschênes  1 , Antonis A Armoundas, Steven P Jones, Gordon F Tomaselli
Affiliations

Post-transcriptional gene silencing of KChIP2 and Navbeta1 in neonatal rat cardiac myocytes reveals a functional association between Na and Ito currents

Isabelle Deschênes et al. J Mol Cell Cardiol. 2008 Sep.

Abstract

The Ca(2+)-independent transient outward potassium current (I(to)) encoded by the Kv4 family of potassium channels, is central to normal repolarization of cardiac myocytes. KChIPs are a group of Ca(2+)-binding accessory subunits that modulate Kv4-encoded currents. However, the biophysical effects of KChIP2 on Kv4 currents raise questions about the role that KChIP2 plays in forming the native I(to). Previous heterologous expression studies demonstrated that the Na channel beta1 subunit modulates the gating properties of Kv4.3 to closely recapitulate native I(to) suggesting that Na(v)beta1 may modulate the function of Kv4-encoded channels in native cardiomyocytes. Therefore we hypothesized the existence of a structural or functional complex between subunits of I(to) and I(Na). In co-immunoprecipitation of proteins from neonatal rat ventricular myocardium (NRVM), Na(v)beta1 was pulled-down by Kv4.x antibodies suggesting a structural association between subunits that comprise I(to) and I(Na). Remarkably, post-transcriptional gene silencing of KChIP2 in NRVM, using small interfering RNAs specific to KChIP2, suppressed both cardiac I(to) and I(Na) consistent with a functional coupling of these channels. KChIP2 silencing suppressed Na channel alpha and beta1 subunit mRNA levels, leaving Kv4.x mRNAs unaltered, but reducing levels of immunoreactive proteins. Post-transcriptional gene silencing of Na(v)beta1 reduced its protein expression. Silencing of Na(v)beta1 also reduced mRNA and protein levels of its alpha-subunit, Na(v)1.5. Surprisingly, silencing of Na(v)beta1 also produced a reduction in KChIP2 mRNA and protein as well as Kv4.x proteins resulting in remarkably decreased I(Na) and I(to). These data are consistent with a novel structural and functional association of I(Na) and I(to) in NRVMs.

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Figures

Figure 1
Figure 1
Co-Immunoprecipitation of potassium and sodium channel subunits (A) Co-Immunoprecipitation of the Navβ1 subunit by Kv4.2 and Kv4.3 antibodies. Lysates from NRVMs transfected with a control non-silencing siRNA were immunoprecipitated with Kv4.x antibodies (IP antibodies). The blot was then hybridized with an anti-Navβ1 antibody and the Kv4.x IP antibodies. The NRVM lysate lanes represent total cell lysates from control cells transfected with a control non-silencing siRNA. The supernatant lanes are the non-bound lysate after incubation with the beads crosslinked with anti-Kv4.2 or Kv4.3 IP antibodies. The IP lanes correspond to the elution from beads crosslinked with either antibodies against Kv4.2 or Kv4.3 respectively and incubated with the protein lysate. The beads + Lysate lanes correspond to non-specific binding of the lysates on beads with no antibody and the Beads no Ab lane represents beads with neither antibody nor lysate. The results demonstrate an interaction between Navβ1 and Kv4.x proteins in NRVM. These are representative blots from a total of five identical experiments. (B) Co-immunoprecipitation in transfected HEK 293 cells with KChIP2 antibody to assess interaction of KChIP2 with Nav1.5 and Kv4.3. Each lane corresponds to a different transfection, the bottom gels are probed with the IP antibody, KChIP2, and the top gels with either Nav1.5 or Kv4.3 antibodies. The IP lanes correspond to the elution from beads crosslinked with KChIP2 antibody and incubated with the protein lysates from HEK 293 cells for the specific transfection indicated in each lane. Lysate lanes correspond to the transfected HEK 293 cell lysates before IP. The supernatant lanes are the non-bound lysate after incubation with the beads crosslinked with anti-KChIP2 antibody. The data demonstrate that Kv4.3, as previously reported, associates with KChIP2, while Nav1.5 does not
Figure 2
Figure 2
Effect of KChIP2 on Nav1.5 biophysical properties. HEK 293 cells were transfected with either Nav1.5 (n=8), Nav1.5 + KChIP2 (n=14), Nav1.5 + Navβ1 (n=12) or Nav1.5 + KChIP2 + Navβ1 (n=10). (A) I/V relationship revealed a significant increased in peak current densities (at voltages −40 to +10 mV) when Nav1.5 was co-expressed with either KChIP2 or Navβ1 and both (p<0.05). Neither steady-state inactivation (B) nor recovery from inactivation (C) were modified.
Figure 3
Figure 3
Effect of KChIP2 siRNA on mRNA and protein levels. (A) Real-time PCR quantification shown in arbitrary units (A.U.) of mRNA transcripts extracted from NRVMs transfected with KChIP2 siRNA normalized to 18S rRNA and then normalized to NRVMs transfected with either a non-silencing control siRNA (black bar) or a single base pair mismatch siRNAs for KChIP2 (gray bar). (B) Representative Western blots of ion channel subunits and of housekeeping proteins for normalization. (C) A bar graph, in arbitrary units (A.U.), of the relative protein density demonstrates a dramatic reduction in KChIP2, Kv4.2, Kv4.3, Nav1.5 and Navβ1 proteins and a more modest reduction in Kv1.4 when normalized to either GAPDH or calsequestrin and then normalized to the quantity of the immunoreactive protein present in NRVMs transfected with the non-silencing control siRNA. RNA and proteins were isolated from at least three different transfections for each condition. * indicate the genes for which the mRNA or protein level was significantly reduced compared to the two control siRNAs (p < 0.05).
Figure 4
Figure 4
Whole cell electrophysiological recordings. Representative action potentials at a 1 Hz stimulation frequency (left panel), representative currents (middle panel) and Na channel family (right panel) obtained from non-transfected NRVMs (A), cells transfected with control non-silencing siRNA (B) and cells transfected with KChIP2 siRNA (C). No action potentials were elicited from NRVMs transfected with KChIP2 siRNA (C, left panel). For currents recording (middle panel), cells were held at −80 mV, stepped to −40 mV (to inactivate INa) followed by voltage steps from −70 to +70 mV in 10 mV increments for 500 msec. Only current records elicited by steps to −70, 0, 30, 50 and 70 mV are shown for clarity. ICa,L is observed with steps to 0 mV in all cell types and Ito at 30, 50 and 70 mV only in the non-transfected cells and cells transfected with control non-silencing siRNA (A and B middle panel). Neither Ito nor INa was detected in NRVMs transfected with KChIP2 siRNA (C, middle panel). The right panel represents a family of sodium currents recorded using the protocols shown in the inset. Low external sodium (25mM) was used for the non-transfected and control siRNA cells in order to ensure voltage clamp control. In presence of the KChIP2 siRNA, physiological external sodium concentrations were used and even when stepping to very negative voltages (−120mV) no sodium currents were elicited. Averaged current-voltage relationships for INa measured with reduced extracellular sodium (except for cells transfected with KChIP2 siRNA) (D) and Ito (E).
Figure 5
Figure 5
Navβ1 silencing (A) Real-time PCR quantification shown in arbitrary units (A.U.) of mRNA transcripts extracted from NRVMs transfected with Navβ1 siRNA normalized to 18S rRNA and then normalized to NRVMs transfected with either a non-silencing control siRNA (black bar) or a single base pair mismatch siRNAs for Navβ1 (grey bar). (B) A bar graph of the relative density of immunoreactive proteins in arbitrary units quantified from Western blots. The relative band intensity was normalized to either GAPDH or calsequestrin for loading and then normalized to the quantity of the immunoreactive proteins present in cells transfected with the non-silencing siRNA control. RNA and Proteins were isolated from at least three different transfections for each condition. * indicate the genes for which the mRNA or protein level was significantly reduced compared to the two control siRNAs (p < 0.05). (C) A two-pulse protocol is used to elicit currents from NRVM transfected with control non-silencing siRNA or Navβ1 siRNA. Cells were held at −80 mV, stepped to −40 mV (to inactivate INa) followed by voltage steps from −70 to +70 mV in 10 mV increments for 500 msec. Only the sodium current present in the inactivating pulse (−40 mV) and the current elicited by the step to +70 mV (Ito) are shown for clarity. These cells had normal IK1 and ICa,L. (D) Sodium current family recorded using the protocol in the inset. In control cells, currents were recorded in presence of low extracellular sodium (25mM), whereas physiological sodium concentration was used in cells transfected with the Navβ1 siRNA. The data confirm a dramatic reduction in sodium currents in presence of the Navβ1 siRNA.
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