Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells (original) (raw)
Related papers
British Journal of Pharmacology, 2005
The A-type voltage-dependent K(+) current (I(A)) has been identified in several types of smooth muscle cells including the pulmonary artery (PA), but little is known about the pharmacological and molecular characteristics of I(A) in human pulmonary arterial smooth muscle cells (hPASMCs). We investigated I(A) expressed in cultured PASMCs isolated from the human main pulmonary artery, using patch-clamp techniques, reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR and immunocytochemical studies. With high EGTA and ATP in the pipette, the outward currents were dominated by a transient K(+) current (I(A)), followed by a relatively small sustained outward current (I(K)). I(A) was inhibited by 4-aminopyridine (4-AP) concentration-dependently, and could be separated pharmacologically into two components by tetraethylammonium (TEA) sensitivity. A component was sensitive to TEA, and the second component was insensitive to TEA. I(A) was inhibited by blood depressing substrate (BDS)-II, a specific blocker of K(V)3.4 subunit, and phrixotoxin-II, a specific blocker of K(V)4.2 and 4.3. Flecainide inhibited I(A) concentration-dependently, but it inhibited it preferentially in the presence of TEA (TEA-insensitive I(A)). Systematic screening of expression of K(V) genes using RT-PCR showed the definite presence of transcripts of the I(A)-encoding genes for K(V)3.4, K(V)4.1, K(V)4.2 and K(V)4.3 as well as the I(K)-encoding genes for K(V)1.1, K(V)1.5 and K(V)2.1. The real-time RT-PCR analysis showed that the relative abundance of the encoding genes of I(A) alpha-subunit and K(V) channel-interacting proteins (KChIPs) was K(V)4.2 > K(V)3.4 > K(V)4.3 (long) > K(V)4.1, and KChIP3 > KChIP2, respectively. The presence of K(V)3.4, K(V)4.2 and K(V)4.3 proteins was also demonstrated by immunocytochemical studies, and confirmed by immunohistochemical staining using intact human PA sections. These results suggest that I(A) in cultured hPASMCs consists of two kinetically and pharmacologically distinct components, probably K(V)3.4 and K(V)4 channels.
Molecular Determinants of Voltage-Gated Potassium Currents in Vascular Smooth Muscle
Cell Biochemistry and Biophysics, 2005
Voltage-gated K + channels (K v) play an important role in regulating contraction of vascular smooth muscle cells (VSMC) through their effects on membrane potential and on voltage-gated Ca 2+ channel activity. K v channels are tetrameric structures consisting of four identical or closely related pore-forming α subunits that may be associated with accessory subunits. More than 30 different gene products that contribute to K v channel complexes have been identified to date, some of which are subject to alternative splicing. Consequently, there is an enormous potential diversity in the molecular composition and properties of possible K v channel complexes. Electrophysiologic measurements of K + currents in VSMC suggest the presence of multiple K v channel assemblies including: (1) rapidly inactivating, 4-aminopyridine-sensitive, (2) slowly inactivating, tetraethylammoniuminsensitive, and (3) noninactivating, tetraethylammonium-sensitive components. Based on electrophysiological and expression studies, it is likely that the latter two components are represented by a heteromultimeric complex of Kv1.2 with either Kv1.4 or Kv1.5 and a Kvβ1 subunit, and by at least Kv2.1, respectively. The identity of the first A-type current component, however, is not clear at this time. The relative abundance of these current components appears to vary in VSMC from different anatomical sites, from animals of different ages, and perhaps in VSMC within specific vascular segments. Expression of numerous K v α and β subunits has been demonstrated in VSMC at both the gene and protein level. However, the number of expressed subunits appears to be much larger than the number of apparent K v current components. It remains unclear if all of these transcripts are expressed in VSMC or in other cell types in the tissue, or if expression patterns are homogenous or heterogeneous in VSMC at a given site. Index Entries: Vascular smooth muscle cell (VSMC); voltage-gated K + channels (K v); Ca 2+ channels.
Cell Biochemistry and Biophysics, 2005
Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studies and are known to play essential roles in excitable cell function, providing an essential counterpoin to the various inward currents that trigger excitability. The serum potassium concentration [K o+] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K+ concentration. As well as changes in serum [K o+], tissue acification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K o+-dependent, and the suggestion has been made that pH and K o+ may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K o+. It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K+ ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K+ channels in the open vs inactivated states of the channels by physiological H+ and K+ concentrations represents an important pathway to control Kv channel function in health and disease.
Cell Biochemistry and Biophysics, 2005
Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studied and are known to play essential roles in excitable cell function, providing an essential counterpoint to the various inward currents that trigger excitability. The serum potassium concentration [K + o ] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K + concentration. As well as changes in serum [K + o ], tissue acidification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K + o -dependent, and the suggestion has been made that pH and K + o may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K + o . It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K + ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K + channels in the open vs inactivated states of the channels by physiological H + and K + concentrations represents an important pathway to control Kv channel function in health and disease.
The Journal of Physiology, 2003
Voltage-gated potassium (K V ) channels represent an important dilator influence in the cerebral circulation, but the composition of these tetrameric ion channels remains unclear. The goals of the present study were to evaluate the contribution of K V 1 family channels to the resting membrane potential and diameter of small rat cerebral arteries, and to identify the a-subunit composition of these channels using patch-clamp, molecular and immunological techniques. Initial studies indicated that 1 mmol l _1 correolide (COR), a specific antagonist of K V 1 channels, depolarized vascular smooth muscle cells (VSMCs) in pressurized (60 mmHg) cerebral arteries from _55 ± 1 mV to _34 ± 1 mV, and reduced the resting diameter from 152 ± 15 mm to 103 ± 20 mm. In patch clamped VSMCs from these arteries, COR-sensitive K V 1 current accounted for 65 % of total outward K V current and was observed at physiological membrane potentials. RT-PCR identified mRNA encoding each of the six classical K V 1 a-subunits, K V 1.1-1.6, in rat cerebral arteries. However, only the K V 1.2 and 1.5 proteins were detected by Western blot. The expression of these proteins in VSMCs was confirmed by immunocytochemistry and co-immunoprecipitation of K V 1.2 and 1.5 from VSMC membranes suggested K V 1.2/1.5 channel assembly. Subsequently, the pharmacological and voltage-sensitive properties of K V 1 current in VSMCs were found to be consistent with a predominant expression of K V 1.2/1.5 heterotetrameric channels. The findings of this study suggest that K V 1.2/1.5 heterotetramers are preferentially expressed in rat cerebral VSMCs, and that these channels contribute to the resting membrane potential and diameter of rat small cerebral arteries.
Potassium channels in vascular smooth muscle: a pathophysiological and pharmacological perspective
Fundamental & Clinical Pharmacology, 2019
Potassium (K +) ion channel activity is an important determinant of vascular tone by regulating cell membrane potential (MP). Activation of K + channels leads to membrane hyperpolarization and subsequently vasodilatation, while inhibition of the channels causes membrane depolarization and then vasoconstriction. So far five distinct types of K + channels have been identified in vascular smooth muscle cells (VSMCs); Ca +2-activated K + channels (BK Ca), voltage-dependent K + channels Accepted Article This article is protected by copyright. All rights reserved. (K V), ATP-sensitive K + channels (K ATP), inward rectifier K + channels (K ir), and tandem-two pore K + channels (K 2 P). The activity and expression of vascular K + channels are changed during major vascular diseases such as hypertension, pulmonary hypertension, hypercholesterolemia, atherosclerosis, and diabetes mellitus. The defective function of K + channels is commonly associated with impaired vascular responses and is likely to become as a result of changes in K + channels during vascular diseases. Increased K + channel function and expression may also help to compensate for increased abnormal vascular tone. There are many pharmacological and genotypic studies which were carried out on the subtypes of K + channels expressed in variable amounts in different vascular beds. Modulation of K + channel activity by molecular approaches and selective drug development may be a novel treatment modality for vascular dysfunction in the future. This review presents the basic properties, physiological functions, pathophysiological and pharmacological roles of the five major classes of K + channels that have been determined in VSMCs.
2006
Objectives: Inhibition of voltage-gated K+ (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs) contributes to the development of hypoxic pulmonary vasoconstriction (HPV). Mitochondria have been proposed as the major oxygen sensing organelles in PASMCs. Although a role for mitochondrial-dependent cellular redox state changes that modulate Kv channels has been proposed, the precise mechanism of the interaction between Kv channels and mitochondria remains unclear. To understand these mechanisms the effect of various mitochondrial inhibitors on Kv channel currents (IKv) were investigated in rat PASMCs and PAs. Comparisons were drawn to the mesenteric circulation. Methods: Patch-clamp technique under different intracellular conditions and in the presence o f a variety o f pharmacological tools. Three key parameters of Ikv were O I assessed; activation, inactivation and Ikv block. Additionally, Mg and Ca fluorescent measurements were performed and whole vessel contractility was assessed using a Mulvany-Halpem myograph. Results: The mitochondrial uncoupler CCCP, and mitochondrial electron transport chain (mETC) inhibitors, rotenone, myxothiazol, antimycin and cyanide, induced similar significant changes in all three Ikv parameters. Antimycin-induced effects, as the most pronounced were studied in detail. It was found that these effects 1) cannot be entirely explained by changes in cellular redox state, 2) are mimicked by the ATP synthase inhibitor oligomycin, 3) were significantly inhibited by cell dialysis with 5 mM Na2ATP, EDTA or with 5 (instead of 0.5) mM MgCl2, whereas intracellular MgATP partially reversed the effect, 4) both CCCP and antimycin caused a significant increase in intracellular Mg2+ (Mg2+i) and 5) hypoxia caused an increase in Mg2+i and a leftward shift in Ikv activation, mimicking the effect of mitochondrial inhibitors. Additionally, the involvement of the Na+-Mg2+ (NME), alongside the Na+-Ca2+ (NCE) and Na+-H+ (NHE) exchangers on 7kv activation and block was evaluated using various extracellular and intracellular conditions. 1) Na+0 removal (to block the exchangers) caused i) a leftward in 7kv activation, ii) a significant increase in the slope of the dependency and iii) a decrease in the maximal whole-cell conductance. 2) Removal of IV o I external Mg , intracellular EDTA and 100 pM amiloride, a putative NME inhibitor, all significantly attenuated Na+0-dependent effects on 7kv. 3) The effects on 7kv activation were significantly attenuated by 3 pM KB-R7943 (a reverse mode inhibitor of the NCE) and extracellular alkalanisation by 0.6 pH unit (the conditions facilitating accumulation I I of intracellular Na), but not by elevated [Ca ]j, intracellular BAPTA, extracellular acidification or by the NHE inhibitor 5-(N-methyl-N-isobutyl) amiloride. Significant differences between pulmonary and mesenteric circulation were found, suggesting specificity of the observed mechanism to the pulmonary circulation. Conclusions: Collectively, these findings suggest the presence of a novel 04mitochondrial-mediated Mg i-dependent mechanism in the regulation of Kv channels in PASMCs, which could be involved in HPV. It also suggests that Kv channel activity 04at physiological membrane potentials in PASMCs chiefly depends on Mg i 04" concentration determined by the balance between the extracellular Mg influx and release and its extrusion by the NME, thus representing a novel regulatory mechanism for Kv channels in PASMCs. Despite some similarities the same overall mechanism
Functional properties of K+ currents in adult mouse ventricular myocytes
The Journal of Physiology, 2004
Although the K + currents expressed in hearts of adult mice have been studied extensively, detailed information concerning their relative sizes and biophysical properties in ventricle and atrium is lacking. Here we describe and validate pharmacological and biophysical methods that can be used to isolate the three main time-and voltage-dependent outward K + currents which modulate action potential repolarization. A Ca 2+-independent transient outward K + current, I to , can be separated from total outward current using an 'inactivating prepulse'. The rapidly activating, slowly inactivating delayed rectifier K + current, I Kur , can be isolated using submillimolar concentrations of 4-aminopyridine (4-AP). The remaining K + current, I ss , can be obtained by combining these two procedures: (i) inactivating I to and (ii) eliminating I Kur by application of low concentration of 4-AP. I ss activates relatively slowly and shows very little inactivation, even during depolarizations lasting several seconds. Our findings also show that the rate of reactivation of I to is more than 20-fold faster than that of I Kur. These results demonstrate that the outward K + currents in mouse ventricles can be separated based on their distinct time and voltage dependence, and different sensitivities to 4-AP. Data obtained at both 22 and 32 • C demonstrate that although the duration of the inactivating prepulse has to be adapted for the recording temperature, this approach for separation of K + current components is also valid at more physiological temperatures. To demonstrate that these methods also allow separation of these K + currents in other cell types, we have applied this same approach to myocytes from mouse atria. Molecular approaches have been used to compare the expression levels of different K + channels in mouse atrium and ventricle. These findings provide new insights into the functional roles of I Kur , I to and I ss during action potential repolarization.
British Journal of Pharmacology, 2006
The aim of this study was to determine the molecular identity of a transient K+ current (termed IUF) in mouse portal vein myocytes using pharmacological and molecular tools. Whole cell currents were recorded using the ruptured patch con from either acutely dispersed single smooth muscle cells from the murine portal vein or human embryonic kidney cells. Reverse transcriptase polymerase reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse portal vein using primers specific for various voltage-dependent K+ channels, auxillary subunits and calcium-binding proteins. Immunocytochemistry was undertaken using an antibody specific for Kv4.3. IUF had a mean amplitude at +40 mV of 558 +/- 50 pA (n = 32) with a mean time to peak at +40 mV of approximately 4 ms. IUF activated and inactivated with a half maximal voltage of -12 +/- 2 mV and -85 +/- 2 mV, respectively. IUF was relatively resistant to 4-aminopyridine (5 mM produced 30 +/- 6 % block at +20 mV) but was inhibited effectively by flecainide (IC50 value was 100 nM) and phrixotoxin II. This pharmacological profile is consistent with IUF being comprised of Kv4.x proteins and this is supported by the results from the quantitative PCR and immunocytochemical experiments. These data represent a rigorous investigation of the molecular basis of vascular transient K+ currents and implicates Kv4.3 as a major component of the channel complex.
2002
A large array of voltage-gated K + channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 Ag/ ml) inhibited the Kv currents by 29.2 F 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 Ag/ml) by 24.5 F 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 Ag/ ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 Ag/ml each) induced an additive inhibition of Kv currents by 42.3 F 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 Ag/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.