An NH2-Terminal Multi-Basic RKR Motif Is Required for the ATP-Dependent Regulation of hIK1

We previously demonstrated that the ATP/PKA?dependent activation of the human intermediate conductance, Ca²⁺?activated K⁺ channel, hIK1, is dependent upon a C?terminal motif. The NH₂?terminus of hIK1 contains a multi?basic ¹³RRRKR¹⁷ motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation (¹⁵RKR¹⁷/AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole?cell patch?clamp. However, cell?surface -immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the ¹⁵RKR¹⁷/AAA mutation we performed inside?out patch clamp recordings where we observed no difference in Ca²⁺ affinity between the wild?type and mutated channels. However, in contrast to wild?type hIK1, channels expressing the ¹⁵RKR¹⁷/AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the ¹⁵RKR¹⁷/AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the ¹⁵RKR¹⁷/AAA mutation resulted in a four?fold lower channel open probability (P_o), in the presence of saturating Ca²⁺ and ATP, compared to wild?type hIK1. In conclusion, these results represent the first demonstration for a role of the NH₂?terminus in the second messenger?dependent regulation of hIK1 and, in -combination with our previous findings, suggest that this regulation is dependent upon a close NH₂/C?terminal association.