Thomas Baukrowitz

Modulation of K+ current by frequency and external [K+]: A tale of two inactivation mechanisms

Voltage-activated K+ currents and their inactivation properties are important for controlling frequency-dependent signaling in neurons and other excitable cells. Two distinct molecular mechanisms for K+ channel inactivation have been described: N-type, which involves rapid occlusion of the open channel by an intracellular tethered blocker, and C-type, which involves a slower change at the extracellular mouth of the pore. We find that frequency-dependent cumulative inactivation of Shaker channels is very sensitive to changes of extracellular [K+] in the physiological range, with much more inactivation at low [K+]out, and that it results from the interaction of N- and C-type inactivation. N-type inactivation enhances C-type inactivation by two mechanisms. First, it inhibits outward K+ flux, which normally fills an external ion site and thus prevents C-type inactivation. Second, it keeps the channels activation gate open even after repolarization, allowing C-type inactivation to occur for a prolonged period.

Use-Dependent Blockers and Exit Rate of the Last Ion from the Multi-Ion Pore of a K+ Channel

Quaternary ammonium blockers inhibit many voltage-activated potassium (K+) channels from the intracellular side. When applied to Drosophila Shaker potassium channels expressed in mammalian cells, these rapidly reversible blockers produced use-dependent inhibition through an unusual mechanism-they promoted an intrinsic conformational change known as C-type inactivation, from which recovery is slow. The blockers did so by cutting off potassium ion flow to a site in the pore, which then emptied at a rate of 105 ions per second. This slow rate probably reflected the departure of the last ion from the multi-ion pore: Permeation of ions (at 107 per second) occurs rapidly because of ion-ion repulsion, but the last ion to leave would experience no such repulsion.

Gating of Ca2+-Activated K+ Channels Controls Fast Inhibitory Synaptic Transmission at Auditory Outer Hair Cells

Fast inhibitory synaptic transmission in the central nervous system is mediated by ionotropic GABA or glycine receptors. Auditory outer hair cells present a unique inhibitory synapse that uses a Ca2+-permeable excitatory acetylcholine receptor to activate a hyperpolarizing potassium current mediated by small conductance calcium-activated potassium (SK) channels. It is shown here that unitary inhibitory postsynaptic currents at this synapse are mediated by SK2 channels and occur rapidly, with rise and decay time constants of approximately 6 ms and approximately 30 ms, respectively. This time course is determined by the Ca2+ gating of SK channels rather than by the changes in intracellular Ca2+. The results demonstrate fast coupling between an excitatory ionotropic neurotransmitter receptor and an inhibitory ion channel and imply rapid, localized changes in subsynaptic calcium levels.

Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle.

For cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels to open, they must be phosphorylated by protein kinase A and then exposed to a hydrolyzable nucleoside triphosphate, such as ATP. To test whether channel opening is linked to ATP hydrolysis, we applied VO4 and BeF3 to CFTR channels in inside-out patches excised from cardiac myocytes. These inorganic phosphate analogs interrupt ATP hydrolysis cycles by binding tightly in place of the released hydrolysis product, inorganic phosphate. The analogs acted only on CFTR channels opened by ATP and locked them open, increasing their mean open time by 2-3 orders of magnitude. These findings establish that opening and closing of CFTR channels are coupled to an ATP hydrolysis cycle.

KCNJ10 gene mutations causing EAST syndrome (epilepsy, ataxia, sensorineural deafness, and tubulopathy) disrupt channel function

Mutations of the KCNJ10 (Kir4.1) K+ channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henles loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.

Inward rectification in KATP channels: a pH switch in the pore.

Inward‐rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage‐dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage‐dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine‐mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward‐rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH–dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C–terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH‐dependent rectification. This pH‐dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH.

Long-chain acyl-CoA esters and phosphatidylinositol phosphates modulate ATP inhibition of KATP channels by the same mechanism

Phosphatidylinositol phosphates (PIPs, e.g. PIP2) and long‐chain acyl‐CoA esters (e.g. oleoyl‐CoA) are potent activators of Katp channels that are thought to link Katp channel activity to the cellular metabolism of PIPs and fatty acids. Here we show that the two types of lipid act by the same mechanism: oleoyl‐CoA potently reduced the ATP sensitivity of cardiac (Kir6.2/SUR2A) and pancreatic (Kir6.2/SUR1) Katp channels in a way very similar to PIP2. Mutations (R54Q, R176A) in the C‐ and N‐terminus of Kir6.2 that greatly reduced the PIP2 modulation of ATP sensitivity likewise reduced the modulation by oleoyl‐CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl‐CoA and PIP2 on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP4‐, ADP3‐ and 2′(3′)‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐monophosphate (TNP‐AMP2‐)) and diadenosine tetraphosphate (Ap4A5‐) ruled out a mechanism where oleoyl‐CoA or PIP2 attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl‐CoA) did not activate but inhibited Katp channels (IC50= 265 ± 33 μM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap4A) are ligands of the inhibitory ATP‐binding site on Kir6.2.

H Bonding at the Helix-Bundle Crossing Controls Gating in Kir Potassium Channels

Summary Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.

Structural and functional analysis of the putative pH sensor in the Kir1.1 (ROMK) potassium channel

The pH‐sensitive renal potassium channel Kir1.1 is important for K+ homeostasis. Disruption of the pH‐sensing mechanism causes type II Bartter syndrome. The pH sensor is thought to be an anomalously titrated lysine residue (K80) that interacts with two arginine residues as part of an ‘RKR triad’. We show that a Kir1.1 orthologue from Fugu rubripes lacks this lysine and yet is still highly pH sensitive, indicating that K80 is not the H+ sensor. Instead, K80 functionally interacts with A177 on transmembrane domain 2 at the ‘helix‐bundle crossing’ and controls the ability of pH‐dependent conformational changes to induce pore closure. Although not required for pH inhibition, K80 is indispensable for the coupling of pH gating to the extracellular K+ concentration, explaining its conservation in most Kir1.1 orthologues. Furthermore, we demonstrate that instead of interacting with K80, the RKR arginine residues form highly conserved inter‐ and intra‐subunit interactions that are important for Kir channel gating and influence pH sensitivity indirectly.

Molecular Basis of Inward Rectification: Polyamine Interaction Sites Located by Combined Channel and Ligand Mutagenesis

Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter.