The Substates with Mutants That Negatively Charged Aspartate in Position 172 Was Replaced with Positive Charge in Murine Inward Rectifier Potassium Channel (Murine Kir2.1)

ISo,I.Ashmole,P.R.Stanfield 대한약리학회 2003 The Korean Journal of Physiology & Pharmacology Vol.7 No.5

Intrinsic Gating in Inward Rectifier Potassium Channels (Kir2.1) with Low Polyamine Affinity Generated by Site Directed Mutagenesis

So, I.,Ashmole, I.,Soh, H.,Park, C.S.,Spencer, P.J.,Leyland, M.,Stanfield, P.R. The Korean Society of Pharmacology 2003 The Korean Journal of Physiology & Pharmacology Vol.7 No.3

We have studied mutant forms of Kir2.1 in which an aspartate residue (D172), important for gating by intracellular polyamines, is replaced by one of three basic residues (Arg, Lys or His). Such channels are highly selective for $K^+$, but show inward rectification that is a shallow function of voltage compared with that found in wild type. This inward rectification occurs with a reduced affinity for spermine and persists in the absence of polyamines. Though the unitary current-voltage relation shows some inward rectification, it is insufficient to account for that seen under whole cell recording. Channels open and shut under single channel recording, and changes of $P_{open}$ appear to generate inward rectification. In D172H, the reduction in affinity for spermine is greater when His is protonated at low $pH_i$. The effective valency for spermine is reduced from $3.09{\pm}0.07$ in wild type to $1.95{\pm}0.09$ in D172H at $pH_i$ 6.3. In the presence of dual mutants of Kir2.1, where E224 is also replaced, spermine affinity becomes undetectable. However, channels still show inward rectification and open and shut under hyper- and depolarisation, respectively. We suggest that Kir2.1 channel are able to undergo conformation changes; these changes may be important physiologically in generating inward rectification, the normal parameters of which are set by the binding of polyamines such as spermine.

The Substates with Mutants That Negatively Charged Aspartate in Position 172 Was Replaced with Positive Charge in Murine Inward Rectifier Potassium Channel (Murine Kir2.1)

So, I.,Ashmole, I.,Stanfield, P.R. The Korean Society of Pharmacology 2003 The Korean Journal of Physiology & Pharmacology Vol.7 No.5

We have investigated the effect on inducing substate(s) of positively charged residues replaced in position 172 of the second transmembrane domain in murine inward rectifier potassium channels, formed by stable or transient transfection of Kir2.1 gene in MEL or CHO cells. Single channel recordings were obtained from either cell-attached patches or inside-out patches excised into solution containing 10 mM EDTA to rule out the effect of $Mg^{2+}$ on the channel gating. The substate(s) could be recorded with all mutants D172H, D172K and D172R. The unitary current-voltage (I-V) relation was not linear with D172H at $pH_i$ 6.3, whereas the unitary I-V relation was linear at $pH_i$ 8.0. The relative occupancy at $S_{LC}$ was increased from 0.018 at $pH_i$ 8.0 to 0.45 at $pH_i$ 5.5. In H-N dimer, that was increased from 0.016 at $pH_i$ 8.0 to 0.23 at $pH_i$ 5.5. The larger the size of the side chain or $pK_a$ with mutants (D172H, D172K and D172R), the more frequent the transitions between the fully open state and substate within an opening. The conductance of the substate also depended upon the pKa or the size of the side chain. The relative occupancy at substate $S_{LC}$ with monomer D172K (0.50) was less than that in K-H dimer (0.83). However, the relative occupancy at substate with D172R (0.79) was similar to that with R-N dimer (0.82). In the contrary to ROMK1, positive charge as well as negative charge in position 172 can induce the substate rather than block the pore in murine Kir2.1. The single channel properties of the mutant, that is, unitary I-V relation, the voltage dependence of the mean open time and relative occupancy of the substates and the increased latency to the first opening, explain the intrinsic gating observed in whole cell recordings.

Intrinsic Gating in Inward Rectifier Potassium Channels (Kir2.1) with Low Polyamine Affinity Generated by Site Directed Mutagenesis

I So,I Ashmole,H Soh,CS Park,PJ Spencer,M Leyland,PR Stanfield 대한생리학회-대한약리학회 2003 The Korean Journal of Physiology & Pharmacology Vol.5 No.1

We have studied mutant forms of Kir2.1 in which an aspartate residue (D172), important for gating by intracellular polyamines, is replaced by one of three basic residues (Arg, Lys or His). Such channels are highly selective for K<SUP>+</SUP>, but show inward rectification that is a shallow function of voltage compared with that found in wild type. This inward rectification occurs with a reduced affinity for spermine and persists in the absence of polyamines. Though the unitary current-voltage relation shows some inward rectification, it is insufficient to account for that seen under whole cell recording. Channels open and shut under single channel recording, and changes of <I>P</I><SUB>open</SUB> appear to generate inward rectification. In D172H, the reduction in affinity for spermine is greater when His is protonated at low pH<SUB>i</SUB>. The effective valency for spermine is reduced from 3.09⁑0.07 in wild type to 1.95⁑0.09 in D172H at pH<SUB>i</SUB> 6.3. In the presence of dual mutants of Kir2.1, where E224 is also replaced, spermine affinity becomes undetectable. However, channels still show inward rectification and open and shut under hyper- and depolarisation, respectively. We suggest that Kir2.1 channel are able to undergo conformation changes; these changes may be important physiologically in generating inward rectification, the normal parameters of which are set by the binding of polyamines such as spermine.

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

이영미,Gareth A. Thompson,Ian Ashmole,Mark Leyland,Insuk So,Peter R. Stanfield 대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba2+ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K+ currents in the absence and presence of externally applied Ba2+. Wild type Kir2.1 was blocked by externally applied Ba2+ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba2+ blockage. The estimated Kd(0) was 108μM for Kir2.1 wild type, 124μM for a concatameric WT-Y145V dimer, 109μM for a WT-Y145L dimer, and 267μM for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba2+, also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba2+ and a decrease in the on transition rate constant (kon). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba2+ blockage in Kir 2.1.

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba<SUP>2+</SUP>

Young Mee Lee,Gareth A. Thompson,Ian Ashmole,Mark Leyland,Insuk So,Peter R. Stanfield 대한생리학회-대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba²⁺ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K⁺ currents in the absence and presence of externally applied Ba²⁺. Wild type Kir2.1 was blocked by externally applied Ba²⁺ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba²⁺ blockage. The estimated K_d(0) was 108ՌM for Kir2.1 wild type, 124ՌM for a concatameric WT-Y145V dimer, 109ՌM for a WT-Y145L dimer, and 267ՌM for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba²⁺, also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba²⁺ and a decrease in the on transition rate constant (k_on). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba²⁺ blockage in Kir 2.1.

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External $Ba^{2+}$

Lee, Young-Mee,Thompson, Gareth A.,Ashmole, Ian,Leyland, Mark,So, In-Suk,Stanfield, Peter R. The Korean Society of Pharmacology 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.1

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on $Ba^{2+}$ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary(CHO) cells and used the whole cell patch-clamp technique to record $K^+$ currents in the absence and presence of externally applied $Ba^{2+}$. Wild type Kir2.1 was blocked by externally applied $Ba^{2+}$ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of $Ba^{2+}$ blockage. The estimated $K_d(0)$ was 108 ${\mu}M$ for Kir2.1 wild type, 124 ${\mu}M$ for a concatameric WT-Y145V dimer, 109 ${\mu}M$ for a WT-Y145L dimer, and 267 ${\mu}M$ for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for $Ba^{2+}$, also showing two components with very different time courses. Mutants D172K and D172R(lining the central, aqueous cavity of the channel) showed both a decreased affinity to $Ba^{2+}$ and a decrease in the on transition rate constant(${\kappa}_{on}$). These results imply that residues stabilising the cytoplasmic end of the selectivity filter(T141, S165) and in the central cavity(D172) are major determinants of high affinity $Ba^{2+}$ blockage in Kir 2.1.