Quinn CC and Begenisich T (2006), Pharmacology and surface electrostatics of the ...

XB-ART-36599

J Membr Biol 2006 Jan 01;2121:51-60. doi: 10.1007/s00232-006-0039-9.

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Pharmacology and surface electrostatics of the K channel outer pore vestibule.

Quinn CC , Begenisich T .

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In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels.

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Species referenced: Xenopus laevis
Genes referenced: kcnb1 kcnc1 kcnc3

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	Figure 1. Sequence and structure of the outer vestibule of K channels. Bottom, Amino acid sequence alignment of three K channels in the region that forms much of the channel pore outer vestibule. In blue are the two lysine residues (K356 and K382) in Kv2.1 channels that are a major focus of this study. Kv2.1 and Kv3.1 channels have a tyrosine amino acid (shown in green) at the position that is important for high-affinity TEA block (see text). In red are several amino acids (aspartate and glutamate) in the vestibule area that have negatively charged side chains. Top left, Structures of gallamine and TEA. Bottom left, Top-view structure of KcsA. The fourfold symmetry of the channel (in ribbon form) is apparent in this view. The Kv2.1 K356 and K382 lysine residues are shown with space-filling atoms (without hydrogens). The tyrosine important for TEA binding is shown with “ball and stick” atoms in green. Right, Side view of the channel with two subunits removed for clarity. Apparent are the outer vestibule and narrow selectivity filter. As in the top view, the relevant lysines and tyrosines are shown with space-filling and ball-and-stick atoms, respectively.
	Figure 2. Ionic strength-dependent TEA and gallamine block of Kv3.1 channels. (A) Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a –80-mV holding voltage. Calibration, 1 μA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from three to five measurements and SEM limits (if larger than symbol) are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.14 and 0.055 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.89 for normal- and low-ionic strength conditions, respectively. (B) Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a –80-mV holding voltage. Calibration, 0.5 μA, 20 ms. Bottom, Dose-response relation for gallamine block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from three to five measurements and SEM limits are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.066 and 0.017 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.94 for normal- and low ionic-strength conditions, respectively.
	Figure 3. Ionic strength-dependent TEA block of Kv2.1 channels. Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a −80-mV holding voltage. Calibration, 2 μA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from 4–10 measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the pooled normal- and low-ionic strength data with Kapp and Bmax values of 5.1 mM and 0.83, respectively.
	Figure 4. Voltage dependence of TEA block of Kv3.1 and Kv2.1 channels. (A) Fraction of Kv3.1 channel current blocked by 100 μM TEA at the indicated membrane potentials in normal- (■) and low- (○) ionic strength solutions. (B) Fraction of Kv2.1 channel current blocked by 5 mM TEA at the indicated membrane potentials in normal- (■) and low- (○) ionic strength solutions.
	Figure 5. Ionic strength-dependent TEA block of mutant Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K382Q channels in normal- (■) and low- (○) ionic strength solutions. Mean values from three measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 4.9 and 2.1 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.96 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K356F channels in normal- (■) and low- (○) ionic strength solutions. Mean values from four to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 6.1 and 2.8 mM for normal- and low- ionic strength conditions, respectively. Bmax values of 0.73 and 0.80 for normal- and low-ionic strength conditions, respectively.
	Figure 6. Ionic strength-dependent TEA and gallamine block of K356F/K382Q Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) in normal- (■) and low- (○) ionic strength solutions. Mean values from three to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 5.6 and 2.8 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.80 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for gallamine block of current (at +40 mV) in normal- (■) and low- (○) ionic strength solutions. Mean values from three to six measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 1.2 and 0.13 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.47 and 0.56 for normal- and low-ionic strength conditions, respectively.
	Figure 7. Electrostatic potential map of the outer vestibule of Kv2.1 channels. Top view of the channel with the pore in the middle; the fourfold symmetry of these K channels is apparent. Electrostatic potentials computed as described in Materials and Methods and mapped onto the surface of the Kv2.1 channel. Negatively charged amino acid side chains are red, and blue represents positive charges. The two lysine residues that are the focus of this work (K336 and K382) as well as the other exposed charged amino acids are indicated in one of the four channel subunits.

References [+] :

Andalib, The external TEA binding site and C-type inactivation in voltage-gated potassium channels. 2004, Pubmed