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B-PO02-015 ENHANCING MUTANT IKSCHANNEL ACTIVITY BY ALTERING ENDOGENOUS PIP2 LEVELS AND ITS INTERACTION WITH PKA SIGNALLING PATHWAY

      Background

      Phosphatidylinositol-4,5-biphosphate (PIP2) is implicated in the regulation and modulation of the IKS channel.

      Objective

      N/A

      Methods

      We initially transfected Human Embryonic Kidney (HEK) cells with a KCNQ1 gene, along with KCNE1 to form the wild type (WT) IKS channel. The cells were also transfected with a constitutively active PI(4)P 5-kinase(PIP5K), which increases endogenous levels of PIP2. To ensure the enzyme remains localised at the plasma membrane we attached it to CFP-FKBP and we co-transfected the cells with cherry tagged Lyn11-FRB construct that tethers to the plasma membrane.

      Results

      We substituted serine with alanine at site 27 and 92(S27A/S92A) to generate a mutant known to disrupt cAMP mediated upregulation, there was a statistical increase in current density when co-expressed with CF-PIP5K. We then substituted serine with aspartic acid (S27D/S92D) to create a Phosphomimetic mutation, this mutant reproduces the effects of sympathetic mediated augmentation of IKS channel. Increased PIP2 levels failed to increase current density in S27D/S92D mutant, implying the channel is at its maximum activity and hence we failed to observe any further modulation.
      In the presence CF-PIP5K, whole cell voltage clamp recordings demonstrated a statistically significant increase in WT channel activity (at +80mV, p<0.001), when compared to unaltered PIP2 conditions. Heterozygous Serine566phe and Phe340del mutants had statistically significant reduction in current density compared to wild type in basal conditions. When these mutants were expressed with the active CF-PIP5K, Serine566phe and Phe340 had an increase current. We then proceeded to interrogate how PIP2 interacts with sympathetic signaling system. Pseudojanin (PJ) causes depletion of PIP2 hence perturbing channel activity. When PJ was expressed with KCNQ1 and KCNE1 we observed an 80% reduction in channel activity at +80mV. When we perfused these cells with isoprenaline the channel activity was restored to normal.

      Conclusion

      Here we illustrate how increasing PIP2 levels can revive IKS channel activity in mutant genotype therefore supporting evidence of its capabilities as a potential therapeutic tool. This modulation is independent of the PKA-cAMP system.