The aim of the proposed research is to evaluate the role of protein kinase A and protein kinase C in the modulation of lymphocyte voltage dependent I+ channels and to determine the consequences of K+ channel modulation on the membrane potential. Experiments will be carried out on CTLL-2 lymphocytes, which lack endogenous voltage-dependent K+ channels, transfected with Kv, 1.3 or Kv3.1 K+ current will be recorded from transfected cells using the conventional whole-cell configuration of patch-clamp as well as perforated-patch and cell-attached patch configurations. Membrane potential will be determined by zero-current potential in current clamp or determined from a slow ramp at hyperpolarized voltages in voltage-clamp. Steady-state and kinetic parameters of the K+ current will be measured under control conditions, and then after activation or inhibition of protein kinase A and C by pre- incubation with appropriate agents. In some experiments cells will be dialyzed with pipette solutions containing purified protein kinase A (PKA) or protein kinase C (PKC) enzyme with or without their appropriate peptide inhibitions. In various patch-clamp configurations the dependence of PKA and PKC effects upon the integrity of the intracellular environment will be tested. Short-and long-term effects as well as temperature dependence of PKC and PKA modulators will also be studied. The physiological consequence of the observed changes in K+ current parameters will be tested when the membrane potential of transfected cells is determined under circumstances similar to those for K+ current measurements. Overall, the project will provide useful information about the significance of protein kinase A and protein kinase C in the regulation of different lymphocyte K+ channels. These studies are important for understanding the function of K+ channels during normal T-cell development, mitogen/antigen activation and autoimmune disorders.
| Panyi, G; Deutsch, C (1996) Assembly and suppression of endogenous Kv1.3 channels in human T cells. J Gen Physiol 107:409-20 |
| Panyi, G; Sheng, Z; Deutsch, C (1995) C-type inactivation of a voltage-gated K+ channel occurs by a cooperative mechanism. Biophys J 69:896-903 |
