SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5

Abstract

The voltage-gated potassium (Kv) channel Kv1.5 mediates the I(Kur) repolarizing current in human atrial myocytes and regulates vascular tone in multiple peripheral vascular beds. Understanding the complex regulation of Kv1.5 function is of substantial interest because it represents a promising pharmacological target for the treatment of atrial fibrillation and hypoxic pulmonary hypertension. Herein we demonstrate that posttranslational modification of Kv1.5 by small ubiquitin-like modifier (SUMO) proteins modulates Kv1.5 function. We have identified two membrane-proximal and highly conserved cytoplasmic sequences in Kv1.5 that conform to established SUMO modification sites in transcription factors. We find that Kv1.5 interacts specifically with the SUMO-conjugating enzyme Ubc9 and is a target for modification by SUMO-1, -2, and -3 in vivo. In addition, purified recombinant Kv1.5 serves as a substrate in a minimal in vitro reconstituted SUMOylation reaction. The SUMO-specific proteases SENP2 and Ulp1 efficiently deconjugate SUMO from Kv1.5 in vivo and in vitro, and disruption of the two identified target motifs results in a loss of the major SUMO-conjugated forms of Kv1.5. In whole-cell patch-clamp electrophysiological studies, loss of Kv1.5 SUMOylation, by either disruption of the conjugation sites or expression of the SUMO protease SENP2, leads to a selective approximately 15-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation. Reversible control of voltage-sensitive channels through SUMOylation constitutes a unique and likely widespread mechanism for adaptive tuning of the electrical excitability of cells.

Fig. 1.

Kv1.5 contains two conserved consensus SUMOylation motifs and interacts with Ubc9. (A) A schematic representation of Kv1.5. T1, tetramerization domain; S1–S6, transmembrane domains. Aligned vertebrate Kv1.5 sequences centered on human K221 (Motif 1) and human K536 (Motif 2) are on the left, and the corresponding regions in human Kv channels are on the right. The core and flanking Gly/Pro residues of the motif are boxed. Asterisks indicate channels with predicted SUMOylation motifs. A SUMOylation consensus is shown above. (B) Structural model of Kv1.5 using the coordinates of Kv1.2-β2 (24). Modeled consensus SUMOylation motifs on each of the three depicted α-subunits are highlighted in red. SUMO-2 is shown for comparison. (C) In vitro interaction between Kv1.5 and Ubc9. Kv1.5 N556 corresponds to a C-terminal truncation lacking the last 57 aa but retaining both SUMOylation motifs. Load corresponds to 10% of applied material.

Fig. 2.

Kv1.5 is SUMO modified in vivo and in vitro. (A) Cos-7 cells were cotransfected as described in Materials and Methods with 200 ng of pCDNA3 Ubc9 and, as indicated, 200 ng of pCDNA3 HA-SUMO3, 200 ng of pCDNA3.1 hKv1.5 V5-His, 160 ng of pCDNA3.1 hKv1.5 N556 V5-His, or an equimolar amount of the corresponding empty vector. His-tagged proteins were purified and detected by immunoblotting with anti-HA (Top) and anti-V5 (Middle and Bottom) antibodies. Middle is an overexposed version of Bottom. SUMO-modified species of Kv1.5 are indicated with arrows, and nonspecific bands are indicated with asterisks. (B) Samples were transfected and processed as in A except cells were harvested in the presence or absence of NEM as indicated, and samples were treated with GST or the SUMO-specific protease GST-Ulp1. Arrows indicate SUMO-modified species of Kv1.5. (C) Purified hKv1.5 was incubated with the indicated SUMO conjugation machinery components as described in Materials and Methods. Arrows indicate SUMO-modified species of Kv1.5.

Fig. 3.

Modification of Kv1.5 by multiple SUMO isoforms requires intact SUMOylation motifs. (A) Cos-7 cells were cotransfected with 3 μg each of pCDNA3 Ubc9, pCDNA3 HA-SUMO1/2/or 3, and 1 μg of pCDNA3.1 hKv1.5 V5-His or an equimolar amount of the corresponding empty vector. Samples were analyzed as in Fig. 2A. Relative exposure times of the anti-HA immunoblots are indicated below each blot. Arrows indicate SUMO-modified species of Kv1.5. (B) Cos-7 cells were transfected with pCDNA3-based vectors for the indicated proteins. Transfection and sample analysis were as in Fig. 2A. SUMO-modified species are indicated with arrows, and nonspecific bands observed occasionally are indicated with an asterisk.

Fig. 4.

Disruption of SUMOylation motifs alters Kv1.5 inactivation. Cos-7 cells were cotransfected with 0.1 μg each of pEGFP-C1 (to identify transfected cells) and a pCDNA3-based vector for the expression of the indicated Kv1.5 variant. Recordings were obtained as described in Materials and Methods. (A) Current–voltage curves. Data represent average ± SEM of 11 WT and 11 K221/536R cells. (B) Voltage dependence of steady-state activation (n = 11 and 10 for WT and K221/536R, respectively). (C) Voltage dependence of steady-state inactivation (n = 7–10 for each construct). The key shows calculated V₅₀ values.

Fig. 5.

SENP2-mediated loss of Kv1.5 SUMOylation alters inactivation. (A) Cos-7 cells were cotransfected as in Fig. 2A with an additional 200 ng of pCMV or pCMV FLAG SENP2 (71–590). Samples were analyzed as in Fig. 2A. Whole-cell lysates were resolved by SDS/PAGE and probed with anti-FLAG antibodies (Lower Right). Arrows indicate SUMO-modified species of Kv1.5 in the anti-HA and anti-V5 immunoblots and SENP2 in the anti-FLAG blot. (B) Cos-7 cells were cotransfected with 0.1 μg of the indicated Kv1.5 plasmid, 0.1 μg of pEGFP-C1, and 0.5 μg of pCMV FLAG SENP2 (71–590). Voltage dependence of steady-state inactivation in the presence of SENP2 was obtained from seven and six cells for WT and K221/536R, respectively. Data in the absence of SENP2 are from Fig. 4C.