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	Figure 1. Mode shift of recombinant human Kv12.1 channels.(A) Representative patch clamp recording of a CHO cell transiently transfected with human Kv12.1 channels activated by the voltage steps as indicated. (B) Voltage protocols consisted of a 200 ms conditioning potential step to −60 mV (black) or 0 mV (blue) followed by 600 ms activating pulse potentials from −140 mV to +10 mV (10 mV increments). Tail currents were elicited either at −120 mV or at 0 mV to minimize time at hyperpolarized membrane potentials (voltage protocols were established in a recent publication [10]). (C-E) Summary of voltage dependence of human Kv12.1 channels. (C) Voltage dependence was analyzed with Boltzmann fits to individual recordings as shown in (A). Solid lines in (C) represent Boltzmann fits to averaged data. Depolarized conditioning potential of 0 mV induced a large shift of voltage dependence to hyperpolarized potentials, as recently reported [10]. (D) shows mean Vh and (E) displays mean slope factor of channel activation in dependence of conditioning potentials of −60 mV and 0 mV (values were derived from Boltzmann fits shown in (C).
	Figure 2. Human Kv12.1 channels are insensitive to GqPCR/PLCβ signaling.(A + B) Activation of muscarinic Gq protein-coupled receptor type 1 (m1R) by Oxo-M (10 µM) strongly inhibited bona fide PI(4,5)P2-sensitive Kv7.4 and Kv7.3T channels. In contrast, Kv12.1-mediated currents were resistant to stimulation of m1R. (A) shows representative Kv7.4- and Kv12.1-mediated whole cell currents before (black) and at the end of a 60 s application of 10 µM Oxo-M (red) (voltage protocol and scale bars as indicated). (B) shows a summary of recordings as presented in (A). (C-E) Steady-state Kv12.1 currents elicited by voltage steps between −140 mV and + 10 mV were not sensitive to activation of m1R through Oxo-M (10 µM). (F) Neither activating kinetics nor (G) deactivating kinetics of Kv12.1 channels were altered upon activation of m1R (time constants were calculated from double-exponential fits to the activating current component and to deactivating tail currents). (C + D) show representative whole cell Kv12.1 currents elicited by the voltage protocols indicated. To induce voltage-dependent mode shift of Kv12.1 channels, voltage protocols consisted of a 200 ms conditioning potential step to (C) −60 mV or (D) 0 mV followed by 600 ms activating pulse potentials from −140 mV to + 10 mV (10 mV increments). (H + I) Oxo-M-dependent activation of m1R did not change voltage-dependence or mode-shift of Kv12.1 channels expressed in CHO cells. (H) shows a summary of the voltage dependence of human Kv12.1 channels and (I) displays mean Vh derived from Boltzmann fits to individual recordings as shown in (C) an (D) (solid lines in (H) represent Boltzmann fits to averaged data; Data with Oxo-M were analyzed at the end of 60 s Oxo-M application; control recordings are also shown in Figure 1). (J + K) In CHO cells coexpressing m1R together with Kv7.4, application of Oxo-M (10 µM) induced robust and reversible translocation of the optical PI(4,5)P2/I(1,4,5)P3 biosensor PLCδ1-PH-mRFP from the membrane to the cytoplasm. PLCδ1-PH-RFP translocation was indistinguishable between cells coexpressing Kv12.1 or Kv7.4 channels together with m1R. Thus, PLCβ activation was comparable in cells expressing Kv7.4 or Kv12.1. (J) shows mean RFP fluorescence intensities measured in confocal sections averaged over a region of interest in the cytoplasm of CHO cells coexpressing PLCδ1-PH-RFP together with either Kv12.1 or Kv7.4. (K) shows representative images of a CHO cell expressing Kv12.1 together with m1R before (top), after 60 s application of Oxo-M (middle) and after wash out of the agonist (bottom). The scale bar represents 10 µm.
	Figure 3. In CHO cells, human Kv12.1 channels are insensitive to activation of a voltage-sensitive PI(4,5)P2/PI(3,4,5)P3 5-phosphatase from ciona intestinalis.(A + B) Activation of Ci-VSP through 30 s depolarization of the holding potential to +80 mV reversibly inhibited recombinant Kv7.2 and Kv7.3T channels. In contrast, Kv12.1-mediated current amplitudes were largely insensitive to voltage-dependent activation of Ci-VSP. (A) shows representative recordings of Kv7.2, Kv7.3T or Kv12.1 currents before (black) and at the end of Ci-VSP activation for 30 s (red) (voltage steps to activate K+ channel were applied every 5 s; Ci-VSP was activated by depolarization to + 80 mV between these voltage steps). (B) displays averaged time course of recordings as shown in (A). (C) In only three out of five cells, we found slight acceleration of Kv12.1 activating kinetics upon voltage-dependent activation of Ci-VSP. (C) shows exemplary normalized Kv12.1 currents activating upon a voltage step from −80 mV to −20 mV before (black) and after activation of Ci-VSP at +80 mV for 30 s (red).

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