THE SENSITIVITY OF THE HUMAN KV1.3 (HKV1.3) LYMPHOCYTE K-293 HOST-CELLS( CHANNEL TO REGULATION BY PKA AND PKC IS PARTIALLY LOST IN HEK)

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-termin al truncated (Delta 459-523) form that lacks the putative PKA Ser(468) phosphorylation site were stably transfected in human embryonic kidne y (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of th e full-length channel whereas clustering was observed in the case of t he truncated channel. Some staining within the cell cytoplasm was foun d in both instances, suggesting an active process of biosynthesis, Ana lyses of the K+ current by the patchclamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding p otential (HP) of -80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to -40 mV and the current amplitude in creased in a voltage-dependent manner. The parameters of activation we re -5.7 and -9.9 mV (slope factor) and -35 mV (half activation, V-0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope f actor) and -17.3 and -39.0 mV (V-0.5) for the full-length and truncate d channels, respectively. The activation time constant of the full-len gth channel for potentials ranging from -30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at -30 mV to 1800 msec at 40 mV. The unit current amplitude meas ured in cells bathing in 140 mM KCl was 1.3 +/- 0.1 pA at 40 mV, the u nit conductance, 34.5 pS and the zero current voltage, 0 mV. Both form s of the channels were inhibited by TEA, 4-AP, Ni2+ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is f ully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 +/ - 7.3% and 38.7 +/- 9.4% inhibition of the full-length and the truncat ed channels, respectively. 8-BrcAMP induced a 39.4 +/- 5.4% inhibition of the full-length channel I,ut had no effect (8.6 +/- 8.3%) on the t runcated channel. Cell dialysis with alkaline phosphatase had no effec ts, suggesting that the decreased sensitivity of the transfected chann els to PKA and PKC was not due to an already phosphorylated channel. P atch extract experiments suggested that the hKv1.3 channel was partial ly sensitive to PKA and PKC. Cotransfecting the KV beta 1.2 subunit re sulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kv beta 2 subunit had no effects. Our resul ts indicate that the hKv1.3 lymphocyte channel retains its electrophys iological characteristics when transfected in the Kv beta-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is sig nificantly reduced.