The role of PSD-95 in the rearrangement of Kv1.3 channels to the immunological synapse

Abstract

Establishment of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells is a key step in the adaptive immune response. Several proteins accumulate in the IS, such as the Kv1.3 potassium channel; however, the mechanism of this translocation is unknown. PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3. Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells. These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.

Protein Kinase A–Phosphorylated K V 1 Channels in PSD95 Signaling Complex Contribute to the Resting Membrane Potential and Diameter of Cerebral Arteries

“…37, 38 However, two recent reports identified PSD95 as a potentially important scaffold of K V 1 channels in lymphocytes and in cVSMCs. 16, 39 First, mutation of the K V 1.3 subunit PDZ binding motif or knockdown of PSD95 in T lymphocytes inhibited the recruitment of K V 1.3 into contact points on the cell surface, implying an important role of PSD95-K V 1 channel interaction in T-cell activation. 39 Second, we demonstrated in cVSMCs that PSD95 is expressed and antisense knockdown of PSD95 in rat CA for 72 hours results in a concomitant loss of K V 1 channel protein and vasodilator function.…”

“…16, 39 First, mutation of the K V 1.3 subunit PDZ binding motif or knockdown of PSD95 in T lymphocytes inhibited the recruitment of K V 1.3 into contact points on the cell surface, implying an important role of PSD95-K V 1 channel interaction in T-cell activation. 39 Second, we demonstrated in cVSMCs that PSD95 is expressed and antisense knockdown of PSD95 in rat CA for 72 hours results in a concomitant loss of K V 1 channel protein and vasodilator function. These findings suggested a critical role of PSD95 in maintaining the expression of K V 1 channels in the cerebral circulation.…”

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome

“…Mutational analysis of the conservative regions would be problematic since it has been demonstrated that simple point mutations of identical parts of Shaker channel can cause nonconducting channels [25,32]. On the contrary, relatively large perturbations in the distal C-terminus of the channel are well tolerated without a significant effect on the gating of Kv1.3 [57]. Therefore, we decided to study a mutant Kv1.3 which lacks the putative cholesterol motifs of the distal C-terminal.…”

“…If CHOL and 7DHC acts on these proposed receptor sites then Kv1.3ΔC should be CHOL and 7DHC insensitive. Previously, we showed that biophysical characteristics of the mGFP-Kv1.3-WT and mGFP-Kv1.3ΔC are identical; removal of the C-terminal did not change significantly the gating properties of Kv1.3 [57]. mGFP-Kv1.3-WT and mGFP-Kv1.3ΔC expressing CHO cells were loaded using 420 μM CHOL and 7DHC, and ionic currents were measured in outside-out patch configuration.…”

“…Cells were passed every 2-3 days. CHO cells were transduced with retroviral particles containing mGFP-Kv1.3-WT and mGFP-Kv1.3ΔC constructs as described in [57].…”