Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel

Potassium (K+) channels are membrane proteins with the remarkable ability to very selectively conduct K+ ions across the membrane. High-resolution structures have revealed that dehydrated K+ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na+ flux in the absence of K+ and introduce significant proton conductance. Consistent with K+ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K+ conditions but undergoes transitions to dilated low-FRET states in high-Na+/low-K+ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K+ and Na+ dependence of SF dynamics to higher K+ and lower Na+ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K+-induced SF configuration versus nonselective dilated conformations

Ligand-induced closure of inward rectifier Kir6.2 channels traps spermine in the pore

Small organic amines block open voltage-gated K(+) channels and can be trapped by subsequent closure. Such studies provide strong evidence for voltage gating occurring at the intracellular end of the channel. We engineered the necessary properties (long block times with unblock kinetics comparable to, or slower than, the kinetics of gating) into spermine-blocked, ATP-gated (N160D,L157C) mutant K(ATP) channels, in order to test the possibility of “blocker trapping” in ligand-gated Kir channels. Spermine block of these channels is very strongly voltage dependent, such that, at positive voltages, the off-rate of spermine is very low. A brief pulse to negative voltages rapidly relieves the block, but no such relief is observed in ATP-closed channels. The results are well fit by a simple kinetic model that assumes no spermine exit from closed channels. The results incontrovertibly demonstrate that spermine is trapped in channels that are closed by ATP, and implicate the M2 helix bundle crossing, or somewhere lower, as the probable location of the gate

Blockade of TRPV channels by intracellular spermine

The Vanilloid thermoTRP (TRPV1-4) subfamily of TRP channels are involved in thermoregulation, osmoregulation, itch and pain perception, (neuro)inflammation and immune response, and tight control of channel activity is required for perception of noxious stimuli and pain. Here we report voltage-dependent modulation of each of human TRPV1, 3, and 4 by the endogenous intracellular polyamine spermine. As in inward rectifier K channels, currents are blocked in a strongly voltage-dependent manner, but, as in cyclic nucleotide-gated channels, the blockade is substantially reduced at more positive voltages, with maximal blockade in the vicinity of zero voltage. A kinetic model of inhibition suggests two independent spermine binding sites with different affinities as well as different degrees of polyamine permeability in TRPV1, 3, and 4. Given that block and relief occur over the physiological voltage range of action potentials, voltage-dependent polyamine block may be a potent modulator of TRPV-dependent excitability in multiple cell types