Molecular Mechanism of Use-Dependent Activation of Kv1.2 Channel Complexes and its Impact on Regulation of Neuronal Excitability

Abstract

Voltage-gated potassium channels (Kv) play a critical role in the regulation of threshold and action potential morphology in virtually all excitable tissues. The Kv1.2 channel in particular plays a significant role in normal neuronal function. Genetic deletion of the Kv1.2 subunit is invariably lethal in mice shortly after birth, and in humans Kv1.2 channelopathies precipitate epileptic encephalopathy and ataxia. We have investigated a poorly recognized regulatory mechanism that profoundly affects Kv1.2 function, termed ‘use-dependent activation’, whereby repetitive stimulation leads to dramatic potentiation of channel current. The physiological significance of this observation is that potentiation of Kv1.2 function may act as a silencing mechanism in repetitively firing neurons. Our findings suggest that use-dependent activation is mediated by an extrinsic regulatory factor that binds Kv1.2 in a state-dependent manner. Point mutations of a putative binding region in the S2-S3 linker suggest important steric and charge requirements to allow interaction of the regulatory factor. While sensitivity is exclusive to Kv1.2 subunits, use-dependent activation can be harnessed in heteromeric channels containing at least one Kv1.2 subunit. We extend these observations by demonstrating that use-dependent activation persists in primary cultured neurons. Using tityustoxin for acute and long-term inhibition of Kv1.2-containing channels in neuronal cultures, we have investigated the role of Kv1.2 subunits on action potential firing and development of neuronal connectivity. Overall, we have dissected the sequence determinants of use-dependent activation of Kv1.2 channels, and made strides toward understanding the implications of this regulatory mechanism in neuronal function.