Novel exon 1 protein‐coding regions N‐terminally extend human KCNE3 and KCNE4

Abstract

The 5 human (h)KCNE b subunits each regulate various cation channels and are linked to inherited cardiac arrhythmias. Reported here are previously undiscovered protein-coding regions in exon 1 of hKCNE3 and hKCNE4 that extend their encoded extracellular domains by 44 and 51 residues, which yields full-length proteins of 147 and 221 residues, respectively. Full-length hKCNE3 and hKCNE4 transcript and protein are expressed in multiple human tissues; for hKCNE4, only the longer protein isoform is detectable. Twoelectrode voltage-clamp electrophysiology revealed that, when coexpressed in Xenopus laevis oocytes with various potassium channels, the newly discovered segment preserved conversion of KCNQ1 by hKCNE3 to a constitutively open channel, but prevented its inhibition of Kv4.2 and KCNQ4. hKCNE4 slowing of Kv4.2 inactivation and positive-shifted steady-state inactivation were also preserved in the longer form. In contrast, full-length hKCNE4 inhibition of KCNQ1 was limited to 40% at +40 mV vs. 80% inhibition by the shorter form, and augmentation of KCNQ4 activity by hKCNE4 was entirely abolished by the additional segment. Among the genome databases analyzed, the longer KCNE3 is confined to primates; full-length KCNE4 is widespread in vertebrates but is notably absent from Mus musculus. Findings highlight unexpected KCNE gene diversity, raise the possibility of dynamic regulation of KCNE partner modulation via splice variation, and suggest that the longer hKCNE3 and hKCNE4 proteins should be adopted in future mechanistic and genetic screening studies.-Abbott, G. W. Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4. FASEB J. 30, 2959-2969(2016. www.fasebj.orgIon channels are complex macromolecules that consist of multiple pore-forming and non-pore-forming subunits in vivo. Voltage-gated potassium (Kv) channels are a numerous and diverse class generated by 40 different a subunits in human tissues that can each come together to form the functional assembly, a tetramer of a subunits. The 40 a subunits are categorized into several subfamilies, and subfamily-specific heterotetramerization commonly occurs, which further diversifies the resultant complexes and the currents they generate (1). In addition, multiple different classes of non-pore-forming (b) subunits, either transmembrane or cytosolic, can coassemble with Kv a subunits to create the considerable complexity required for normal cellular functions of higher animals (2).The most widely studied group of transmembrane Kv b subunits are the KCNE proteins, also referred to as MinK-related peptides (3, 4), with .1000 published studies on this gene family to date. KCNE proteins are so widely studied because they can exert sometimes dramatic effects on Kv channel function (5-11), are obligate partners for many Kv a subunits in vivo (5, 6, 9-17), can alter Kv channel pharmacology with respect to clinically relevant and investigational drugs (18,19), and their reach is impressive, each modulating multiple Kv a subunits and, in some c...

Regulation of human cardiac potassium channels by full-length KCNE3 and KCNE4

“…The reported migration patterns of the two forms of mature KCNE4 protein on SDS-PAGE gels correspond to ~25 kDa for KCNE4S41 and ~30 kDa for KCNE4L40. Here, an antibody raised to a peptide sequence present on only the long form of KCNE4 detected a ~30 kDa band in CHO cells expressing KCNE4L, but no bands in the 20–40 kDa size range in CHO cells expressing KCNE4S.…”

“…1A,B). In support of their potential relevance to human cardiac function, transcripts coding for KCNE3L and KCNE4L were previously detected in human atrial tissue40.…”

“…After transfer, PVDF membranes were blocked in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBST) and 5% dried milk for 1 h at room temperature, washed 3 × 5 minutes in TBST, and then incubated overnight at 4 °C with rabbit polyclonal primary antibodies in TBST containing 5% (w/v) dried milk. α-KCNE4L, a custom antibody (Genscript) raised to a peptide based on the KCNE4L-specific N-terminal segment (residues 17–32), sequence RAQSRTEQKNPLGLD-C (1/1000 dilutions)40 was used to probe for KCNE4L. Blots were next washed in TBST (3 × 5 minutes), incubated for 1 hour at room temperature with 1/5000 Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Bio-Rad) in TBST containing 1% (w/v) dried milk, and then washed again in TBST (3 × 5 minutes) before chemiluminescent detection using Luminata Forte chemiluminescence substrate (Millipore, Temecula, CA), and analysis using a Gbox system and Gbox software (Syngene).…”

“…For Chinese hamster ovary (CHO) cell biochemistry, CHO cells were transfected with cDNAs encoding human KCNE4S in pCINeo, or human KCNE4L in pCDNA3.1+40. Two days post-transfection, cells were lysed in MiRP buffer, rotated end-over-end in Eppendorf tubes for 2 hours at 4 °C, then centrifuged (4000 ×  g ; 10 min; 4 °C), and the resulting supernatants SDS-PAGE separated, western blotted and analyzed as described for heart samples above.…”

“…Recently, I discovered exon 1 coding regions that generate longer (L) isoforms of human KCNE3 and KCNE4, detected both proteins in various epithelial tissues, and conducted an initial examination of their functional effects40. Here, I demonstrate the expression of KCNE4L protein in human heart, and describe the functional effects of KCNE3L and KCNE4L on the major human cardiac Kv channel α subunits.…”