A New Approach to Study Non-conducting Kv2 Channels

Abstract

Kv2.1 is the most abundant mammalian potassium channel in the body and underlies the majority of delayed rectifier current in central neurons. In addition to its role in action potential regulation, Kv2.1 forms large, stable junctions between the endoplasmic reticulum and plasma membrane, thus giving rise to its striking clustered localization pattern in many cell types. These ER-PM junctions are mediated by an interaction between Kv2.1 and ER VAPs, and this interaction is dynamically regulated by neuronal excitability and insult via calcium-dependent mechanisms. Tens-of-thousands of Kv2.1 channels are expressed on the neuronal surface in order to facilitate the interaction with ER VAPs, however, only a fraction of these channels contribute to whole-cell currents. Instead, the majority of surface Kv2.1 channels are non-conducting, likely to avoid electrically silencing the neuron. These observations suggest that Kv2.1 is uniquely able to perform both structural and electrical functions for the cells in which it is expressed. Although the molecular mechanism of channel silencing remains elusive, our work suggests a relationship between the spatial density of Kv2.1 channels in the plasma membrane and the fraction of channels that conduct, with high channel densities favoring the non-conducting population. In this work, a new combination of optical and electrophysiological approaches is developed in HEK293 cells to quantify conducting and non-conducting channels simultaneously in the same cell. We find that all surface Kv2.1 channels have functional voltage-sensing domains and yet as few as 10% of those channels conduct potassium during strong depolarization. We go on to use this approach to probe regulated conductance for the first time in the second Kv2 isoform, Kv2.2, and in Kv2 channels co-assembled with the auxiliary subunit AMIGO1.