For the MA, K(v) protein expression and current components between WKYs and SHRs were qualitatively consistent, but differences in gene and protein expression were not closely correlated. The higher expression of K(v) subunits in small mesenteric arteries (SMAs) of SHR would tend to maintain normal myogenic activity and vasoconstrictor reserve, and could be viewed as a form of homeostatic remodeling.
Multiple Kv channel complexes contribute to total Kv current in numerous cell types and usually subserve different physiological functions. Identifying the complete compliment of functional Kv channel subunits in cells is a prerequisite to understanding regulatory function. It was the goal of this work to determine the complete Kv subunit compliment that contribute to functional Kv currents in rat small mesenteric artery (SMA) myocytes as a prelude to studying channel regulation. Using RNA prepared from freshly dispersed myocytes, high levels of Kv1.2, 1.5 and 2.1 and lower levels of Kv7.4 α-subunit expression were demonstrated by quantitative PCR and confirmed by Western blotting. Selective inhibitors correolide (Kv1; COR), stromatoxin (Kv2.1; ScTx) and linopiridine (Kv7.4; LINO) decreased Kv current at +40mV in SMA by 46 ± 4%, 48 ± 4% and 6.5 ± 2%, respectively, and Kv current in SMA was insensitive to α-dendrotoxin. Contractions of SMA segments pretreated with 100 nmol/L phenylephrine were enhanced 27 ± 3%, 30 ± 8% and 7 ± 3% of the response to 120 mmol/L KCl by COR, ScTX and LINO, respectively. The presence of Kv6.1, 9.3, β1.1, and β1.2 was demonstrated by RT-PCR using myocyte RNA with expression of Kvβ1.2 and Kv9.3 about 10-fold higher than Kvβ1.1 and Kv6.1, respectively. Selective inhibitors of Kv1.3, 3.4, 4.1 and 4.3 channels also found at the RNA and/or protein level had no significant effect on Kv current or contraction. These results suggest that Kv current in rat SMA myocytes are dominated equally by two major components consisting of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels along with a smaller contribution from Kv7.4 channels but differences in voltage dependence of activation allows all three to provide significant contributions to SMA function at physiological voltages.
The lower abundance of exon15Δ73 transcripts in SHR results in a larger fraction of total Cav1.2 mRNA coding for full-length CaV protein, and the higher abundance of exon9* transcripts and CaVβ3a protein likely contribute to differences in gating and kinetics of CaV currents in SHR. Functional studies of Ca2+ currents in native SMA myocytes and HEK cells transiently transfected with CaV subunits support these conclusions.
We have shown that three components contribute to functional voltage gated K (K ) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K currents in small mesenteric artery myocytes with those of K subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested K subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.
Alternative splicing of the voltage-gated Ca(2+) (CaV) α1-subunit adds to the functional diversity of Ca(2+) channels. A variant with a 73-nt deletion in exon 15 of the Cav1.2 α1-subunit (Cav1.2Δ73) produced by alternative splicing that predicts a truncated protein has been described, but its function, if any, is unknown. We sought to determine if, by analogy to other truncated CaV α1-subunits, Cav1.2Δ73 acts as an inhibitor of wild-type Cav1.2 currents. HEK-293 cells were transfected with Cav1.2Δ73 in a pIRES vector with CD8 or in pcDNA3.1 with a V5/his COOH-terminal tag plus β2 and α2δ1 accessory subunits and pEGFP. Production of Cav1.2Δ73 protein was confirmed by Western blotting and immunofluorescence. Voltage-clamp studies revealed the absence of functional channels in transfected cells. In contrast, cells transfected with full-length Cav1.2 plus accessory subunits and pEGFP exhibited robust Ca(2+) currents. A7r5 cells exhibited endogenous Cav1.2-based currents that were greatly reduced (>80%) without a change in voltage-dependent activation when transfected with Cav1.2Δ73-IRES-CD8 compared with empty vector or pIRES-CD8 controls. Transfection of A7r5 cells with an analogous Cav2.3Δ73-IRES-CD8 had no effect on Ca(2+) currents. Immunofluorescence showed intracellular, but not plasma membrane, localization of Cav1.2Δ73-V5/his, as well as colocalization with an endoplasmic reticulum marker, ER Organelle Lights. Expression of Cav1.2Δ73 α1-subunits in A7r5 cells inhibits endogenous Cav1.2 currents. The fact that this variant arises naturally by alternative splicing raises the possibility that it may represent a physiological mechanism to modulate Cav1.2 functional activity.
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