Kv2.1 channels have important conducting and nonconducting functions and are regulated by ‘silent’ Kv subunits. One of these is Kv9.1, which is co-expressed with Kv2 channels in sensory neurons, and a common allele which changes Ile489 to Val in human Kv9.1 promotes pain sensitivity. Here we examined the effects of Kv9.1 variants on Kv2 channels expressed in HeLa cells and primary hippocampal neurons. Both Kv9.1Ile and Kv9.1Val were retained within the endoplasmic reticulum when expressed individually, but when co-expressed with either Kv2.1 or Kv2.2 they co-localized with Kv2 within surface clusters. Both variants reduced surface expression of Kv2.1 channels and the size of channel clusters, with Kv9.1Val producing a greater reduction in surface expression in both HeLas and neurons. They both caused a similar hyperpolarizing shift in the voltage-dependence of channel activation and inactivation. Concatamers of Kv2.1 and Kv9.1, suggest both a 3:1 and 2:2 ratio of Kv2.1 to Kv9.1 are permitted, although 2:2 resulted in lower surface expression and function. The AMP kinase activator A769662 produced a strongly hyperpolarizing shift in the voltage-dependence of Kv2.1 and Kv2.1-Kv9.1 channel currents. Onset and recovery kinetics of A769662 were rapid and mutational analysis indicated differences in the regulation of hKv2.1 compared to rKv2.1, with a lack of dependence upon Ser444. In conclusion, the Ile489Val substitution in Kv9.1 did not disrupt its association with Kv2 channels nor its effects on channel gating but it did produce a greater reduction in the Kv2.1 surface expression, suggesting that this underlies its association with pain hypersensitivity.